Figure 5.

Circ-Calm4 sponges miR-124-3p to mediate pulmonary arterial smooth muscle cell (PASMC) pyroptosis. A and B, Knockdown of endogenous miR-124-3p by AMO-124-3p abrogated the antipyroptotic effects of Circ-calm4 silencing by Circ-calm4-small-interfering RNA (siRNA) upon hypoxia exposure in PASMCs, as indicated by the 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; n=6). C, Cotransfection with circ-Calm4 siRNA and AMO-124-3p abrogated the antipyroptotic effects on PASMCs upon hypoxia exposure, as indicated by LDH (lactate dehydrogenase) activity (n=6). D, Knockdown of circ-Calm4 by siRNA reduced the positive propidium iodide (PI) staining induced by hypoxia in cells, whereas AMO-124-3p reversed the decrease in PI-positive PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). E, Circ-Calm4 knockdown blocked the fluorescence intensity of Caspase-1 and IL-18 under hypoxia exposure, and this decrease was abrogated after transfection of AMO-124-3p. 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. *P<0.05, **P<0.01, ***P<0.001.