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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. 6 — Loss of cFLIP_L by Usp27x_L expression requires TRIM28, which is required for pIC induced apoptosis and deficiency of Usp27x_L stabilizes cFLIP_L in 293FT cells. A, B, hUsp27x_L induction decreases cFLIP_L protein levels in a TRIM28 dependent manner. GFP-hUsp27x_L doxycycline-inducible WM1158 (no KO) or same cells where the TRIM28 locus had been targeted by CRISPR/Cas9 to generate TRIM28 deficient cells (TRIM28-KO) were stimulated as indicated, lysed in Laemmli buffer and protein levels were determined by Western blot (n = 4). B, Quantification of cFLIP_L protein levels detected by Western blotting (A) after 48 h dox treatment normalized to GAPDH. Shown are the cFLIP_L protein levels compared to the untreated respective cell line (**: p value < 0.005, error bars represent SEM; n = 4). C, TRIM28 is needed for pIC induced cell death. Same cells as in (A) were stimulated as indicated, harvested, fixed and stained for active caspase-3 followed by FACS analyses. Data (means, n = 7; error bars represent SEM). *, **, ***: significant, see 2 section. D, Usp27x- or TRIM28 deficiency does not change endogenous cFLIP level and TRIM28 deficiency does not change Usp27x level (and vice versa) in WM1158 cells. Whole-cell-lysates from control (CTRL), polyclonal Usp27x-deficient (Usp27x-2 and Usp27x-3), or TRIM28-deficient (TRIM28-KO) WM1158 melanoma were run on SDS-gels and endogenous level of TRIM28, Usp27x (using custom-made Usp27x antibody) and cFLIP level were detected by Western blotting. Arrow-heads indicate the specific signal for human Usp27x_L, asterisks indicate unspecific signal (n = 3). E, Usp27x-deficiency does not protect WM1158 cells from pIC-induced apoptosis. WM1158 cells were stimulated with pIC as indicated and apoptosis was measured as described in C (n = 3, Data (means, ns: not significant)). Error bars represent SEM. F, deficiency of Usp27x_L stabilises cFLIP_L in 293FT cells. Same 293FT wild-type (WT) or Usp27x-deficient 293FT cells as described in Figs. 1B and 5D were analysed for endogenous cFLIP level (n = 4). Quantification of cFLIP_L protein levels detected by Western blotting normalized to GAPDH is indicated. Shown are the relative cFLIP_L protein levels (%) compared to the respective WT cell line (p value < 0.05, ± represent SEM; n = 4)