Fig. 3. MCC950 (100 µmol/l) treatment reduces pyroptosis, indicated by lower levels of active Caspase 1 (p20), higher levels of pro-Caspase 1, elevated uncleaved GSDMD as well as reduced cytotoxic N-terminal GSDMD.

a Representative pro-Caspase 1 (p45) (green), active Caspase 1 (p20) (red), and DAPI (blue) immunofluorescence stainings of bEnd5 after 24 h OGD (3.0% O₂, 5.0% CO₂, 95% humidity, 37.0 °C, 1 g/l glucose, red) either with vehicle or with MCC950 treatment. In all, ×20 objective; scale bar = 25 µm. b Top: representative Western Blot depicting the amount of expressed pro-Caspase 1 (45 kDa) in bEnd5 lysates. To control protein loading β-actin was used (expected mass ~42 kDa). Bottom: ratio of pro-Caspase 1 (p45) protein band intensity to actin as loading control after 0 h, 5 h, 10 h, and 24 h of OGD with vehicle or MCC950 (100 µmol/l) treatment (n = 5 out of three independent experiments). c, d Top: representative immunofluorescence stainings of either uncleaved GSDMD or GSDMD (N-terminal) (green). Bottom: either uncleaved GSDMD or GSDMD (N-terminal) intensity of bEnd5 cell cultures after 24 h OGD with either vehicle or MCC950 (100 µmol/l) treatment (n = 9 out of four independent experiments). In all, ×40 objective; scale bar = 5 µm. e, f Top: representative Western Blot depicting the amount of expressed uncleaved (52 kDa) or cleaved (N-terminal) (32 kDa) GSDMD in bEnd5 lysates. To control protein loading β-actin was used (expected mass ~42 kDa). Bottom: ratio of either uncleaved GSDMD or cleaved (N-terminal) GSDMD protein band intensity to actin as loading control after 0 h, 5 h, 10 h, and 24 h of OGD with vehicle or MCC950 (100 µmol/l) treatment (n = 9 or five out of three independent experiments). Data were analyzed by paired t test. *p < 0.05; **p < 0.01.