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. 2019 Aug 30;11(9):1276. doi: 10.3390/cancers11091276

Figure 3.

Figure 3

PC3-derived EVs induce caspase-1 activation and IL-1β maturation in PNT2 cells.PNT2 cells were treated with 100 µg/mL of PC3-derived EVs (PC3-EVs) either in the presence or in the absence of 10 µg/mL LPS + 5 mM ATP (LPS + ATP). After a 24 h exposure, (A) NLRP3, caspase-1 full length and mature caspase-1-p20 expression and (B) pro-IL-1β full length and mature IL-1β-p20 expression were evaluated in total extract analyzed by Western blotting. β-actin was used as a loading control. The images are representative of one out of at least three separate experiments. (C) After a 3 h exposure, p-NF-κB (p65) expression was evaluated in total extract and analyzed by Western blotting. β-actin was used as a loading control. The images are representative of one out of at least three separate experiments. (D) qRT-PCR of IL-1β gene after a 6 h exposure. Gene expression values were normalized to HPRT and presented as 2−ΔΔCt. Relative mRNA gene abundance in PNT2 cells was assumed as 1 (Ctr). Data represent mean ± SD (n = 5). * p < 0.05 vs. PNT2 cells. (E) PNT2 cells were treated with 100 µg/mL of PNT2-derived EVs (PNT2-EVs) for 24 h and caspase-1 full length and mature caspase-1-p20 expression was evaluated in total extract analyzed by Western blotting. β-actin was used as a loading control. The images are representative of one out of n = 5 separate experiments. Numbers below the images represent the ratio between the respective protein and β-actin band intensity.