Research Paper Volume 15, Issue 23 pp 13680—13692

Figure 6. APN suppresses human NP cell pyroptosis by up-regulating miR-135a-5p expression. (A) Detection of miR-135a-5p expression in normal and degenerated human NP tissue samples using qRT-PCR (n=10). (B) Human NP cells were treated with PBS or LPS for 12 h and then transfected with or without lentiviral vector overexpressing APN for an additional 24 h. The expression of miR-135a-5p was analyzed by qRT-PCR (n=3). (C–G) LPS-stimulated human NP cells were transfected with or without miR-135a-5p inhibitor for 24 h, followed by transduction with APN-expressing lentivirus for another 24 h (n=3). (C) TXNIP expression was measured using qRT-PCR and western blot. (D) Western blot was applied to determine the protein levels of NLRP3, caspase-1 and GSDMD-N. (E) Detection of IL-1β and IL-18 levels in the cell culture supernatants using ELISA. (F) The LDH assay kit was utilized to detect LDH activity in the cell culture supernatants. (G) Representative images of fluorescent staining with Hoechst 33342 (blue) and PI (red) and quantitative analysis of PI positive cells. The results are shown as the mean ± SD. **P < 0.01, ***P < 0.001, ^#P < 0.05.