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. 2020 Nov 3;11:5387. doi: 10.1038/s41467-020-19170-5

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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. 3 — a Western blots on extracts from stated cells treated ± IFN-β for 24 h. N = 2 independent blots with one representative blot shown. b 293 cells were co-transduced with shControl or shMPP8 plus shMAVS or a control vector of the same backbone (hygromycin) and GFP was measured on day 6 (left). MAVS depletion was verified using polyI:C, added 24 h before FACS (middle). Summary data shown for experiments in ISRE-GFP and IFN-β-GFP reporter cells (right, n = 3 biologically independent samples). P = 0.0372 (one-tailed paired t test). MFI: mean fluorescent intensity. c Left: qRT-PCR expression of endogenous ISGs in the stated shRNA-treated 293 cell lines at day 6 (GAPDH normalized). N = 4 biological independent experiments with technical duplicates shown. CCL5 and CXCL10 were measured in all experiments while the other 3 ISGs were measured in 3 of the experiments. One-tailed paired t tests showed expression of all ISGs was significantly lower in MDA5−/− cells and double knockout (DKO) cells than in wildtype (WT) controls. IFIT1: p = 0.0063 (MDA5−/−), p = 0.0026 (DKO); IFIT2: 0.0007 (MDA5−/−), 0.0019 (DKO); CCL5: 0.0002 (MDA5−/−), 0.0028 (DKO); CXCL10: 0.0001 (MDA5−/−), 0.0007 (DKO); MxA: 0.0427 (MDA5−/−), 0.0423 (DKO). Expression of CCL5, CXCL10, and MxA was significantly decreased in RIG-I−/− cells compared to controls. CCL5: p = 0.0183; CXCL10: p = 0.0372; MxA: p = 0.0139. Right: MPP8 depletion efficiency summary (n = 4 biologically independent experiments with technical duplicates shown. d Western blots on extracts from cell lines from c treated ± IFN-β for 24 h. N = 2 independent blots with one representative blot shown. e Left: stated knockout or control (WT) THP-1 reporter cell lines were treated with shRNAs and assayed for secreted Lucia luciferase (driven by IFIT-2) 6 days later. N = 4 biologically independent experiments. Luciferase readings were normalized to background values in shControl-treated cells. P = 0.0003 (one-tailed paired t test). Right: western blots on extracts from stated cells. N = 3 independent blots with one representative blot shown. f 293 cells were treated with shRNAs and fixed 6 days later for intracellular staining with dsRNA antibodies, J2/K1 or control antibodies. N = 3 biologically independent samples. Two-tailed unpaired t test p values = 0.0166 (shMPP8 vs. shControl for K1 Ab); 0.0048 (shMPP8 vs. shControl for J2 Ab); 0.0212 (shMPP8 + J2 Ab ± dsRNase). All data presented in this figure show mean values ± SD.