Figure 1.

TYK2 P760L-transduced primary bone marrow cells and factor-free colony formation. (A) Schematic illustration of combinations of kinase-activating germline and kinase-inactivating mutations: TYK2 consists of a four-point-one, ezrin, radixin, moesin (FERM), an atypical Src-homology 2 (SH2), a JAK homology (JH)2 and a JH1 domain. In wild-type (WT) TYK2 the JH2 pseudokinase domain inhibits the JH1 kinase domain at basal state; the JH1 domain is activated upon cytokine receptor engagement (WT, grey). P760L alters the structure of a conserved motif in the JH2 domain which is predicted to attenuate the kinase inhibitory JH2-JH1 interaction (ON, orange). M978F inactivates the JH1 domain (= OFF, blue). Combination of M978F with P760L abolishes the TYK2 kinase activity. (B) Representative colony pictures of transduced bone marrow (BM) cells, taken with 4-fold magnification objective for single colonies. Scale bars show 500 µm. White arrows indicate visible colonies. (C) Replating experiment of transduced BM cells in factor-free methylcellulose (n=3, duplicates, two-way ANOVA with log transformed data). (D) Percentage of Lin^-, Sca-1⁺, c-Kit⁺ (LSK) cells among GFP⁺ TYK2 P760L-transduced BM cells after the first round in methylcellulose (n=3). Cells were gated on single cells, living, GFP⁺, Lin^-, c-Kit⁺ and Sca-1⁺. Representative plots are shown. **P<0.01, ***P<0.001.