Figure 1.

Circ-Calm4 small-interfering RNA (siRNA) inhibits pyroptosis in pulmonary artery smooth muscle cells (PASMCs). A and B, Circ-Calm4 siRNA reversed the increased protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β) induced by hypoxia in PASMCs (n=6). C, Circ-Calm4 siRNA reversed the increased LDH (lactate dehydrogenase) activity in PASMCs subjected to hypoxia for 24 h. LDH release was evaluated with an LDH release kit (n=6). D, Knockdown of circ-Calm4 decreased the number of pyroptotic cells in PASMCs exposed to hypoxia. Cells were detected with annexin V-FITC/propidium iodide (PI) double staining using quantitative fluorescence-activated cell sorting (FACS) analysis. E, Knockdown of circ-Calm4 by siRNA reduced the positive PI staining induced by hypoxia in PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). F, Circ-calm4 siRNA attenuated the fluorescence staining of Caspase-1 and IL-18 induced by hypoxia in PASMCs. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. The graph A-ASC and Caspase-1 were analyzed by the Kruskal-Wallis test followed by Dunn post-test. *P<0.05, **P<0.01, ***P<0.001.