Fig. 1: Costimulation of TLR2, TLR4, and TLR7/8 induces ROS production, GSDMD cleavage and IL-1β hypersecretion. | Cell Death & Disease

Fig. 1: Costimulation of TLR2, TLR4, and TLR7/8 induces ROS production, GSDMD cleavage and IL-1β hypersecretion.

From: Progressive waves of IL-1β release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways

Fig. 1

a Intracellular ROS quantified by H2DCF-DA staining in monocytes unstimulated or stimulated for 1 h as indicated. N = 3. Data are expressed as RFU. b, c Monocytes from healthy donors were incubated with LPS alone or with LPS, R848, Zymosan (LRZ) for the indicated times (hours). (b) Time course of IL-1β secretion, quantified by ELISA. N = 6. Data are expressed as ng/ml (mean ± SEM). (c) One representative Western blot analysis (out of 3 performed) of intracellular and extracellular IL-1β at various times of exposure to LPS or LRZ. d One representative Western blot (out of 5 performed) showing GSDMD cleavage in cell lysates from monocytes cultured 6 h untreated (Unt) or with LRZ or LPS. Arrowhead indicates the p30 N-terminal domain of GSDMD. GAPDH served as loading control. e Secretion of IL-1β by monocytes stimulated with LRZ or LPS in the presence or absence of DPI (20 μM) or As2O3 (1 μM) as indicated. Data are expressed as the percent of IL-1β secreted by LPS or LRZ stimulated monocytes exposed to DPI or As2O3 relative to secretion without drugs. f, g Western blot analyses of GSDMD cleavage in monocytes stimulated 6 h with LRZ or LPS in the presence or absence of DPI (20 μM) or As2O3 as indicated. f One representative western blot out of 3 performed; arrowheads indicate p30 GSDMD. g Ratio of GSDMD p30/GAPDH obtained by quantitative densitometry of the 3 western blots like the one shown in f (mean ± SEM). h, i Western blot analyses of GSDMD cleavage in monocytes stimulated 6 h with LRZ or LPS in the presence or absence of Ac-YVAD, Z-LEVD or Ac-YVAD plus Z-LEVD (Y + L). h One representative western blot out of 3 performed; i Ratio of GSDMD p30 /GAPDH obtained by quantitative densitometry of the 3 western blots like the one shown in h (mean ± SEM). j Caspase dependency of IL-1β secretion. Monocytes were stimulated with LPS or LRZ in the presence or absence of Ac-YVAD, Z-LEVD or Ac-YVAD plus Z-LEVD (Y + L) and analyzed as above. Data are expressed as in e. Data information: One-way ANOVA (a, b, h, i) or Unpaired t-test (e, g) were used for significance (*P < 0.05, **P < 0.01, ***P < 0.001)

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