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. 2019 Jun 19;9:8742. doi: 10.1038/s41598-019-45009-1

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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(A) ATP assay was performed using the Intestinal Epithelial cells (IEC-6) cell supernatants for the groups Control, DETA-NONOate treated, DETA-NONOate treated+ FBA and DETA-NONOate treated + DMPO, to study the concentration of ATP per well. (n = 3, analysis from three samples) (*p < 0.05). Intestinal epithelial (IEC-6) cell line was used as control (CONTROL), one group was treated with the supernatant of IEC-6 cell line that was treated with NO-donor (DETA-NONOate), another group of cells were blocked by FBA and then was treated with the NO-donor (DETA-NONOate) (DETA-NONOate + FBA). The cell groups were used for immunofluorescence imaging. (B) (i–iii) Immunofluorescence images depicting NLRP3 (red)-ASC2 (green) colocalization and (C) (iv–vi) NLRP3 (red)- Caspase-1 (green) colocalization, counterstained with DAPI (blue) in CONTROL, DETA-NONOate,DETA-NONOate + FBA, group of IEC-6 cells. Images were taken at 20X magnification. (D) Morphometric analysis of NLRP3-ASC2 and (E) NLRP3-Caspase1 colocalized events. Y-axis shows % positive immunoreactive area (% ROI) (n = 5, analysis from five separate microscopic fields) (*p < 0.05).