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. 2022 Jan 19;27(1-2):112–132. doi: 10.1007/s10495-021-01706-9

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© The Author(s) 2022

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Induction of hUsp27x_L in WM1158 and 1205Lu leads to loss of cFLIP_L protein and processing of caspase-8 when treated with pIC, but does not alter NF-κB or type I interferon signalling

A, B hUsp27x_L induction decreases cFLIP_L protein levels and increases caspase-8 processing upon pIC treatment. GFP-hUsp27x_L doxycycline-inducible WM1158 (A) were pre-stimulated 48 h ± Dox to induce Usp27x_L and afterwards QVD [10 µM] ± pIC was added for the indicated time points. 1205Lu cells inducibly expressing GFP-hUsp27x_L (B) were stimulated for 48 h ± Dox, ± pIC at the same time as indicated. Cells were lysed in urea lysis buffer and proteins were analyzed by Western blot (for A, B: n = 3). C Quantification of cFLIP_L protein levels detected by Western blotting (A, B) after 48 h dox treatment normalized to GAPDH. Shown are the cFLIP_L protein levels relative to the untreated control (*: p value < 0.05, error bars represent SEM; n = 3). D Overexpression of hUsp27x_L does not alter cFLIP_L mRNA level. GFP-hUsp27x_L doxycycline-inducible WM1158 and 1205Lu cells were treated or not with Dox the day after seeding and harvested at the indicated time points. The mRNA levels of cFLIP_L and house-keeping genes (HPRT and GAPDH) were measured by qPCR. Obtained values were normalized to the respective uninduced cell-line. Error bars represent the geometric SD (ns: adjusted p value > 0.05); *: statistically significant = p-value < 0.05 (n = 3). E The loss of cFLIP_L protein levels due to Usp27x overexpression is partially blocked by the addition of the proteasome inhibitor MG132. Western blot of WM1158-TetR-GFP-hUsp27x_L cells unstimulated or pre-stimulated with Dox for 48 h to induce GFP-hUsp27x_L, followed by ± treatment with MG132 [10 µM, 4 h]. cFLIP_L protein levels were normalized to their GAPDH loading control and quantified against the untreated cells set to 100% (n = 2). F, B, Induction of hUsp27x_L in WM1158 (F) or 1205Lu (B) cells does not alter the levels of NF-κB signal transduction proteins after pIC treatment. GFP-hUsp27x_L doxycycline-inducible WM1158 cells were treated as described for A (n = 3). G Induction of hUsp27x_L in WM1158 cells does not influence the TRL3 induced type I interferon signalling pathway under the same experimental setup used in (A, F). No change in TBK1, phospho-TBK1 nor phospho-IRF3 protein levels were detected, as well as total levels of upstream TRAF3 and TRIF (n = 3)