Figures & data
Figure 1. The cytomorphology of HL-1 cells and AQP4 expression with AQP4 inhibition
(a). Phase contrast images of HL-1 cells without (Sham) and with AQP4i (AQP4i) treatment in the indicated groups. Scale bar = 30 μm. (b). Ultrastructure images of HL-1 cells by utilizing electron microscopy without (Sham) and with AQP4i (AQP4i) treatment in the indicated groups. Scale bar = 100 μm. (c). Western-blotting assay of Aquaporin 4 (AQP4) and GAPDG in HL-1 cells in the aforementioned groups. (d). Quantitative analysis of Aquaporin 4 (AQP4) protein expression in the aforementioned groups. **, P < 0.01; NS, not significant.
Figure 2. The influence of AQP4 inhibition upon cellular viability of HL-1 cells
(a). The cell viability of HL-1 cells without (Sham) and with AQP4i (AQP4i) treatment in the indicated groups. *, P < 0.05; NS, not significant. (b,c). Evaluation of the inhibitory effect of AQP4i to the apoptosis of HL-1 cells by immunofluorescence-based TUNEL intensity assay. Scale bar = 50 μm. D-E. FCM diagram (d) and statistical analysis (e) of the subpopulations of cell cycle in the aforementioned groups. *, P < 0.05; **, P < 0.01; NS, not significant.
Figure 3. The alleviative effect of AQP4i upon ischemia-reperfusion was apoptosis-independent
(a,b). FCM diagram (A) and statistical analysis (B) of the apoptotic HL-1 cells in the aforementioned groups without (Sham) and with AQP4i (AQP4i) treatment. *, P < 0.05; **, P < 0.01. C-D. FCM diagram (c) and statistical analysis (d) of the JC-1 positive cells in the aforementioned groups. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. E-F. The pyroptosis-associated Caspase 1 activity (e) and the concentrations of cytokines including IL-1β and IL-8 (f) in the supernatant of HL-1 cells in the aforementioned groups. *, P < 0.05; **, P < 0.01; NS, not significant.
Figure 4. The inhibitory effect of AQP4i upon pyroptosis in HL-1 cells
(a). Quantitative analysis of the pyroptosis-associated biomarker expression in HL-1 cells without (Sham) and with AQP4i (AQP4i) treatment by utilizing qRT-PCR. *, P < 0.05; **, P < 0.01; NS, not significant. (b). Western-blotting assay of the pyroptosis-associated biomarker and GAPDG expression in HL-1 cells in the aforementioned groups. GAPDH was used as a loading control. (c). Quantitative analysis of the pyroptosis-associated biomarker expression in the aforementioned groups. *, P < 0.05; **, P < 0.01; NS, not significant. (d). Immunofluorescent staining of the pyroptosis-associated biomarker GSDMD expression in HL-1 cells in the aforementioned groups. DAPI was used for nuclear staining. Scale bar = 100 μm.
