Figure 7: Endocytosis of ITO-NPs by PMA-activated THP-1 cells with NLRP3 or ASC knockdown does not elicit pyroptosis but rather apoptosis.

(a) LDH release capabilities as measured in PMA-activated THP-1 either wild-type (WT) or NLRP3 knockdown (def-NLRP3), or ASC knockdown (def-ASC) on exposure to increasing doses of ITO-NPs. (b) PMA-activated THP-1 WT cells exposed to increasing doses of ITO-NPs but not cells with NLRP3 knockdown or ASC knockdown released IL-1β dose-dependently. (c) Similarly, PMA-activated THP-1 WT cells exposed to increasing doses of ITO-NPs but not cells with NLRP3 knockdown or ASC knockdown released TNF-α dose-dependently. (d) PMA-activated THP-1 WT cells or with NLRP3 knockdown or ASC knockdown exposed to ITO-NPs were assessed for cell viability by trypan blue dye exclusion cell count on light microscope. Results are given as number of cells alive and dead cells (×10⁶) with or without exposure to ITO-NPs. (e) PMA-activated THP-1 WT cells or cells with NLRP3 knockdown or ASC knockdown exposed to ITO-NPs were stained with Annexin V and PI to determine the relative number of single positive cells Annexin V⁺PI⁻ (apoptotic cells) compared to the number of cells double positive Annexin V⁺PI⁺ (pyroptotic cells). Results are given as a percentage of total cells with or without exposure to ITO-NPs. (f) TEM analysis of PMA-activated THP-1 WT cells exposed to ITO-NPs exhibited almost exclusively endocytic cells containing ITO-NPs with plasma membrane being ruptured; black arrow shows ITO-NPs being released after plasma membrane has dislocated (red arrow). By contrast, PMA-activated THP-1 cells with NLRP3 knockdown or ASC knockdown exhibited cells with capabilities of ITO-NPs uptake (black arrow), showing morphological features of apoptosis, such as membrane blebs and apoptotic bodies (blue arrow). TEM original magnification x3000 and x8000; 1 representative image is shown in at least 3. Concentration of ITO-NPs was 500 μg/mL, and time of exposure was 16 h, unless stated otherwise. (a–f) n = 3–8 ± SEM, *p < 0.05. TEM abbreviations, Cyto = cytoplasm, N = nucleus and E = endosome.