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. 2020 Nov 20;11:5919. doi: 10.1038/s41467-020-19670-4

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© The Author(s) 2020

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. 6 — a Pulse-chase assay of IL-10-induced STAT3 phosphorylation in differentiated THP1 cells. The indicated strains (F: endogenous full-length ATG16L1; N: CRISPR/Cas9-induced ATG16L1 depletion (guide 1025–1044) plus restoration with the Nt domain, aminoacids 1–299) were treated with PMA (125 ng/ml; 48 h), pre-incubated with IL-10 (50 ng/ml; 30 min on ice), incubated at 37 °C for the shown times and lysed to obtain nuclear extracts for the indicated Western-blots. Corrected densitometric quantifications are shown at the bottom of the Western-blot images. b Quantitative PCR to assess IL-10-induced suppression of TNF and IL-6 mRNA upregulation by LPS in THP1 cells. Cells were pre-activated as in a, treated with LPS (TNF, 10 ng/ml; IL-6, 10 or 60 ng/ml) for 5 h with or without IL-10 (50 ng/ml) and lysed for qPCR. Shown are average fold inhibition values induced by IL-10 −/+ s.d. (TNF: n = 7, IL-6: n = 6 experimental points; *P < 0.05; **P < 0.01, two-sided Student’s t-test). c Pulse-chase assay of IL-10-induced STAT3 phosphorylation in BMDMs. BMDMs from 3 randomly-chosen mouse pairs (F: full-length ATG16L1; N: E230 mice expressing the Nt domain) were pre-incubated with IL-10 (50 ng/ml; 30 min on ice), incubated at 37 °C for the shown times and lysed to obtain nuclear extracts for the indicated Western-blots. Corrected densitometric quantifications are shown at the bottom of the Western-blot images. A statistical analysis of nuclear P-STAT3 signals is shown in Supplementary Fig. 16c. d Quantitative PCR to evaluate IL-10-induced expression of Bcl3. BMDMs from the indicated mice were treated with IL-10 (50 ng/ml, 4 h) and lysed for qPCR. Shown are average fold induction values induced by IL-10 −/+ s.d. (n = 6 mice; *P < 0.05, two-sided Student’s t-test). e Quantitative PCR to assess IL-10-induced suppression of TNF mRNA upregulation by LPS. BMDMs from the indicated mice were treated with LPS (1 ng/ml; 6 h) with or without IL-10 (50 ng/ml) and lysed for qPCR. Shown are average fold inhibition values induced by IL-10 −/+ s.d. (n = 5 mice; *P < 0.05, two-sided Student’s t-test).