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. 2020 May 14;9(5):1216. doi: 10.3390/cells9051216

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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ICMT/Ras-mediated inflammatory responses are regulated by the TIR domain of MyD88 and TRIF. ((A,C), left panel) AP-1-mediated luciferase activity was analyzed by reporter gene assay in HEK293 cells transfected with MyD88 and its mutant constructs [MyD88 (Y58F), MyD88 (ΔITAM), MyD88 (ΔDD), MyD88 (ΔID), and MyD88 (ΔTIR)] (A) and in HEK293 cells transfected with TRIF and its mutant construct CFP-TRIF-ΔTIR ((C), left panel). Luminescence levels were determined with a luminometer. ((B,C), right panel) Total and phospho-protein levels of MAPKs (ERK1/2, JNK1/2, and p38) in HEK293 cells transfected with MyD88 and its mutant constructs [MyD88 (Y58F), MyD88 (ΔITAM), MyD88 (ΔDD), MyD88 (ΔID), and MyD88 (ΔTIR)] (B) and in HEK293 cells transfected with TRIF and its mutant construct CFP-TRIF-ΔTIR ((C), right panel). ((D–G), left panel) Binding of endogenous Ras (D,F) and Myc-tagged RAS and its mutants (Myc-K-RAS-AAA_54-57 and Myc-K-RAS-C185A) (E) to MyD88 ((D,E), left panel), TRIF ((D,E), right panel), and c-Raf ((G), left panel) was evaluated using immunoprecipitation and immunoblotting analyses with total lysates of HEK293 cells transfected with MyD88-WT or MyD88-ΔTIR ((D,E), left panel) and TRIF and TRIF-ΔTIR ((D,E), right panel) in the absence (D,E) or presence ((F,G), left panel) of CyM (30 µM). ((G), right panel) Total and phospho-protein levels of ICMT, c-Raf, ERK1/2, IRAK4, and TRAF6 from whole lysates of HEK293 cells transfected with Myc-tagged RAS or its mutants (Myc-K-RAS-AAA_54–57 and Myc-K-RAS-C185A) were analyzed using immunoblotting analysis. (H) The mRNA expression levels of TIR-domains containing adaptor molecules (MyD88, TRIF, TIRAP, and TRAM) (H, left panel) and inflammatory genes (IL-1β, IL-6, and TNF-α) ((H), right panel) were measured using real-time PCR in MDA-MB-231 cells transfected with Ras in the presence or absence of siTIR (20 nM). **: p < 0.01 and ***: p < 0.001 compared to MyD88 or TRIF-WT-transfected group.

ICMT/Ras-mediated inflammatory responses are regulated by the TIR domain of MyD88 and TRIF. ((A,C), left panel) AP-1-mediated luciferase activity was analyzed by reporter gene assay in HEK293 cells transfected with MyD88 and its mutant constructs [MyD88 (Y58F), MyD88 (ΔITAM), MyD88 (ΔDD), MyD88 (ΔID), and MyD88 (ΔTIR)] (A) and in HEK293 cells transfected with TRIF and its mutant construct CFP-TRIF-ΔTIR ((C), left panel). Luminescence levels were determined with a luminometer. ((B,C), right panel) Total and phospho-protein levels of MAPKs (ERK1/2, JNK1/2, and p38) in HEK293 cells transfected with MyD88 and its mutant constructs [MyD88 (Y58F), MyD88 (ΔITAM), MyD88 (ΔDD), MyD88 (ΔID), and MyD88 (ΔTIR)] (B) and in HEK293 cells transfected with TRIF and its mutant construct CFP-TRIF-ΔTIR ((C), right panel). ((D–G), left panel) Binding of endogenous Ras (D,F) and Myc-tagged RAS and its mutants (Myc-K-RAS-AAA_54-57 and Myc-K-RAS-C185A) (E) to MyD88 ((D,E), left panel), TRIF ((D,E), right panel), and c-Raf ((G), left panel) was evaluated using immunoprecipitation and immunoblotting analyses with total lysates of HEK293 cells transfected with MyD88-WT or MyD88-ΔTIR ((D,E), left panel) and TRIF and TRIF-ΔTIR ((D,E), right panel) in the absence (D,E) or presence ((F,G), left panel) of CyM (30 µM). ((G), right panel) Total and phospho-protein levels of ICMT, c-Raf, ERK1/2, IRAK4, and TRAF6 from whole lysates of HEK293 cells transfected with Myc-tagged RAS or its mutants (Myc-K-RAS-AAA_54–57 and Myc-K-RAS-C185A) were analyzed using immunoblotting analysis. (H) The mRNA expression levels of TIR-domains containing adaptor molecules (MyD88, TRIF, TIRAP, and TRAM) (H, left panel) and inflammatory genes (IL-1β, IL-6, and TNF-α) ((H), right panel) were measured using real-time PCR in MDA-MB-231 cells transfected with Ras in the presence or absence of siTIR (20 nM). **: p < 0.01 and ***: p < 0.001 compared to MyD88 or TRIF-WT-transfected group.