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. 2021 Jan 6;12(1):49. doi: 10.1038/s41419-020-03335-7

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© The Author(s) 2021

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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Fig. 1 — A, B ELISA analysis of cytokines in culture supernatants of BMDMs. LPS-primed or unprimed BMDMs were treated with the indicated concentrations of gefitinib for 8 h. IL-1β (A) or TNF-α (B) release were analyzed by ELISA. C ELISA analysis of cytokines in culture supernatants of THP-1 cells. PMA-differentiated THP-1 cells were treated with the indicated concentrations of gefitinib for 8 h. IL-1β release was analyzed by ELISA. D, E Immunoblot analysis of IL-1β in culture supernatants of BMDMs. LPS-primed BMDMs were treated with the indicated concentrations of gefitinib for 8 h (D), or treated with 20 μM gefitinib for indicated periods (E). Cell-free supernatants (Sup) and cell lysates were subjected to immunoblotting with the indicated antibodies. F, G Immunoblot analysis of IL-1β in culture supernatants of THP-1 cells. PMA-differentiated THP-1 cells were treated with the indicated concentrations of gefitinib for 8 h (F), or treated with 20 μM gefitinib for the indicated periods (G). Cell-free supernatants (Sup) and cell lysates were subjected to immunoblotting with the indicated antibodies. H, I The mRNA expression levels of IL-1β. BMDMs were treated with the indicated concentrations of gefitinib for 8 h or 100 ng/ml LPS for 6 h (H), or treated with 20 μM gefitinib for indicated periods or 100 ng/ml LPS for 6 h (I). The mRNA levels of IL-1β were analyzed by quantitative real-time PCR (normalized with GAPDH mRNA levels). Graphs are shown as mean ± S.D. (n = 3). Statistical significance was determined by student’s t-test; *p < 0.05, **p < 0.01. J, K The mRNA expression levels of IL-1β. THP-1 cells were treated with the indicated concentrations of gefitinib for 8 h or 100 nM PMA for 4 h (J), or treated with 20 μM gefitinib for indicated periods or 100 nM PMA for 4 h (K). The mRNA levels of IL-1β were analyzed by quantitative real-time PCR (normalized with GAPDH mRNA levels). Graphs are shown as mean ± S.D. (n = 3). Statistical significance was determined by student’s t-test; ***p < 0.001. L ASC oligomerisation assay in THP-1 cells. PMA-differentiated THP-1 cells were treated with 20 μM gefitinib for 8 h. DSS-mediated crosslinked pellets (crosslinked-pellets) and soluble lysates (Input) were subjected to immunoblotting with anti-ASC antibody. All data in Fig. 1 are representatives of at least three independent experiments.