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. 2021 Mar 31;11:622727. doi: 10.3389/fonc.2021.622727

Figure 4.

Figure 4

Downregulation of hsa_circ_0001836 induced pyroptosis in glioma cells via epigenetically upregulating NLRP1. (A) U251MG cells were transfected with hsa_circ_0001836 siRNA3 for 72 h. DNA methylation of the NLRP1 promoter region was analyzed by MSP. “U” and “M” represents unmethylated and methylated signal intensity, respectively. The product length of the methylated primers of NLRP1 is 125 bp; and the product length of the unmethylated primers of NLRP1 is 127 bp. (B) The level of NLRP1 in U251MG cells transfected with NLRP1 siRNA1, NLRP1 siRNA2 or NLRP3 siRNA3 analyzed by RT-qPCR. (C) U251MG cells were transfected with hsa_circ_0001836 siRNA3 or co-transfected with hsa_circ_0001836 siRNA3 and NLRP1 siRNA2 for 72 h. CCK-8 assay was applied to determine cell viability. (D) Cell death was detected by flow cytometry. (E) Dying cells revealed by SEM. (F, G) Western blot analysis of NLRP1, GSDMD-N levels in U251MG cells. The relative expressions of NLRP1, GSDMD-N in U251MG cells normalized to β-actin. *P < 0.05, **P < 0.01 vs. siRNA-ctrl group; ##P < 0.01 vs. hsa_circ_0001836 siRNA3 group.