Fig. 6

Inhibiting autophagic flux promotes caspase-4 inflammasome activation. A, B Expression and quantification of beclin1, LC3, p62, caspase-4 and IL-1β in CMECs cultured under normoxia or subjected to H/R (2/2 h) with or without 3-MA (5 mM), VX-765 (10 μM). Data are expressed as mean ± SD (n = 3). ^*P < 0.05 vs. control group, ^**P < 0.05 vs. 3-MA + H/R group, ^***P < 0.05 vs. VX-765 + H/R group. C Analysis of LDH release in CMECs subjected to H/R (2/2 h) ± 3-MA (5 mM) or VX-765 (10 μM). Data are expressed as mean ± SD (n = 5). ^*P < 0.05 vs. control group, ^**P < 0.05 vs. 3-MA + H/R group, ^***P < 0.05 vs. VX-765 + H/R group. D, E Expression and quantification of LC3, p62, caspase-4 and GSDMD in CMECs cultured under normoxia or subjected to H/R (2/2 h) with or without VX-765 (10 µM). Data are expressed as mean ± SD (n = 3). ^*P < 0.05 vs. control group, ^**P < 0.05 vs. control + VX-765 group, ^***P < 0.05 vs. H/R + VX-765 group. F CMECs were cultured for 24 h after transfection with RFP-GFP-LC3. CMECs carring RFP-GFP-LC3 subjected to H/R were treated with or without 3-MA (5 mM), Baf (50 nM) or VX-765 (10 µM). Representative immunofluorescence images of CMECs expressing RFP-GFP-LC3; cell nuclei were stained with DAPI (blue). Red punta represent autophagosomes; yellow punta in merged picture represent autolysosomes. G Semi-quantitative analysis of autophagosomes and autolysosomes in each group. H, I Expression and quantification of LC3, p62 and caspase-4 in CMECs cultured under normoxia or subjected to H/R (2/2 h) with or without Baf (50 nM). Data are expressed as mean ± SD (n = 3). ^*P < 0.05 vs. control group, ^#P < 0.05 vs. control + Baf group, ^##P < 0.05 vs. H/R + Baf group