Fig. 7.

Inhibition of autophagy abrogates the protective effect of estrogen supplementation on pyroptosis without negatively affecting ERα expression. (A) Representative western blots and relative quantitative analysis of Beclin1, LC3B, and SQSTM1 with or without 12 h of application of 60 nM Hcy to HUVECs in the presence or absence of 10 nM E₂ or/and 10 μM 3-MA. (B) Representative western blots and relative quantitative analysis of NLRP3, cleaved caspase 1, and GSDMD with or without 12 h of application of 60 nM Hcy to HUVECs in the presence or absence of 10 nM E₂ or/and 10 μM 3-MA. (C) Representative western blots and relative quantitative analysis of IL-1β and IL-18 with or without 12 h of application of 60 nM Hcy to HUVECs in the presence or absence of 10 nM E₂ or/and 10 μM 3-MA. (D) After transfection of HUVECs with GFP-LC3 adenovirus, immunofluorescence images of cells were taken with or without 60 nM Hcy for 12 h and in the presence or absence of 10 nM E₂ or/and 10 μM 3-MA. DAPI was used to stain the nucleus. Mean optical density analysis of GFP was performed. Scale bar: 50 μm. (E) Immunofluorescence images of cells were taken with or without 60 nM Hcy for 12 h and in the presence or absence of 10 nM E₂ or/and 10 μM 3-MA. Green: GSDMD, red: ERα. DAPI was used to stain the nucleus. Mean optical density analysis of GSDMD and ERα was performed. Scale bar: 50 μm. (F) Immunofluorescence images of cells were taken with or without 60 nM Hcy for 12 h and in the presence or absence of 10 nM E₂ or/and 10 μM 3-MA. Red: Caspase-1. DAPI was used to stain the nucleus. Mean optical density analysis of Caspase-1 was performed. Scale bar: 50 μm. In all experiments, n = 6. **P < 0.01.