Journal: The Journal of Clinical Investigation

Article Title: Clemastine fumarate accelerates accumulation of disability in progressive multiple sclerosis by enhancing pyroptosis

doi: 10.1172/JCI183941

Figure Lengend Snippet: The THP-1 cells were primed with 200 ng/mL LPS overnight and then treated either with medium + DMSO (negative control, Ctrl), 10 μg/mL CLM, 2 mM ATP ± 10 μg/mL CLM, or 2.5 μM nigericin (positive control) for 3 hours, 6 hours, and 18 hours. Release of the proinflammatory cytokine IL-1β into the culture medium of THP-1 cells ( A ) and THP-1 GSDMD –/– cells ( B ), determined by ELISA assay. Activity of caspase-1 in the culture medium of THP-1 cells ( C ) and THP-1 GSDMD –/– cells ( D ), determined using bioluminescent assay. Lytic cell death of THP-1 cells ( E ) and THP-1 GSDMD –/– cells ( F ), determined by LDH activity released into the culture medium. Cell viability of THP-1 cells ( G ) and THP-1 GSDMD –/– cells ( H ) evaluated by the MTS assay. ( A – H ) Data are presented as mean ± SD of 3 independent experiments, each performed in duplicate ( n = 6). One-way ANOVA followed by Dunnett’s multiple-comparisons test was used to compare the testing groups with control group (Ctrl). One-way ANOVA test followed by Holm-Šidák’s multiple comparison was used to compare ATP and ATP+CLM group (to assess the potentiation effect of CLM). * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Panel A , ATP+CLM at 3 hours ( n = 1), nigericin at 3 hours ( n = 3), 6 hours ( n = 5), and 18 hours ( n = 5) — above detection limit of the assay. Panel B , Ctrl at 3 hours ( n = 5) and 6 hours ( n = 3) — below detection limit of the assay. Nigericin at 18 hours ( n = 6) — above detection limit of the assay. ( I ) Flow cytometry analysis of cell uptake of SYTOX Green in THP-1 cells primed with 200 ng/mL LPS overnight and then treated either with medium + DMSO (control), 10 μg/mL CLM, or 2 mM ATP ± 10 μg/mL CLM for 90 minutes. Representative plot of 2 independent experiments.

Article Snippet: The membranes were blocked with 2.5%–5% BSA and then incubated overnight at 4°C with the following primary antibodies: GSDMD rabbit PolyAb (1:1000; Proteintech, 20770-1-AP, lot:00156359), caspase-1/p20/p10 rabbit PolyAb (1:1000; Proteintech, 22915-1-AP, Lot: 00154445) or β-actin antibody (C4), mouse monoclonal IgG1 (1:5000; Santa Cruz Biotechnology Inc., sc-47778/C4).

Techniques: Negative Control, Positive Control, Enzyme-linked Immunosorbent Assay, Activity Assay, MTS Assay, Control, Comparison, Flow Cytometry