Journal: Nature genetics

Article Title: The mutational landscape of cutaneous T-cell lymphoma and Sézary syndrome

doi: 10.1038/ng.3442

Figure Lengend Snippet: Somatic mutations in Sézary syndrome and CTCL. Schematics of TP53, epigenetic factors TET2, BRD9, MLL3, MLL2, CREBBP, SMARCA4 and CHD3 and the CARD11, BRAF, MAPK1, PREX2, cGK1β, JAK3, SH2B3 and STAT3 signaling proteins showing mutations identified in this study. Black and blue circles indicate amino acid substitutions; red circles truncating mutations. Cys, cysteine rich domain; DHSBH, double-stranded beta helix fold domain; Bromo, bromo domain; PHD, Plant-homeodomain; HMG, High mobility group domain; FYRN “FY-rich” domain N-terminal; FYRC “FY-rich” domain C-terminal; SET, Su(var), Enhancer of zest, and Trithorax; Z, zinc finger domain; KIX, binding site of CREB; HAT, histone acetyl transferase domain; Q, glutamine-rich domain; QLQ, Gln, Leu, Gln motif; HAS, helicase/SANT-associated domain; BRK, Brahma and Kismet domain, SNF2_N, SF2 family N-terminal domain; Hel_C, Helicase superfamily C-terminal domain; SnAC, Snf2-ATP coupling, chromatin- remodeling complex; CC-coiled-coil domain; SIM, SUMO interacting domain; CARD, caspase recruitment domain; PDZ, PSD-95 (95 kDa protein involved in signaling in the post-synaptic density), Drosophila disc large tumor suppressor (Dlg1), Zonula occludens-1 protein (zo-1); SH3, Src homology 3 domain; GUK, guanylate kinase domain; TAD, transactivation domain; P rich, proline rich domain; TD, tetradimerization domain; NR, negative regulation domain; RBD, Ras binding domain; C1; protein kinase C-conserved region 1 domain; DH, DBL homology domain; PH, plekstrin homology domain; DEP domain, Dishevelled, Egl-10 and Pleckstrin domain; DIM/LZ, dimerization and leucine zipper domain; IS, auto-inhibitory domain; cGMP, cGMP binding domain; ATP, ATP binding domain; SB, substrate binding domain; FERM, 4.1protein/Ezrin/Radixin/Moesin domain; SH2, Src homology 2 domain; Phe-ZIP, phenylalanine zipper.

Article Snippet: We transfected the lentiviral constructs pLX304-blasV5 CARD11 wild type, CARD11 S615F, E626K, D230N, L215P, M183L and the empty vector with pCMV ΔR8.91 and pMD.G VSVG in HEK293T cells using Fugene 6 (Promega), according the manufacturers protocol.

Techniques: Binding Assay

Journal: Nature genetics

Article Title: The mutational landscape of cutaneous T-cell lymphoma and Sézary syndrome

doi: 10.1038/ng.3442

Figure Lengend Snippet: Functional characterization of Sézary syndrome CARD11 linker domain mutations. (a) Schematic representation of the structure of the CARD11 protein. CARD11 mutations identified are indicated with circles. (b) NFκB luciferase reporter activity in HEK293-T cells transfected with increasing amounts of V5-CARD11 wild-type, S615F and E626K or empty vector. Data is representative of 3 independent experiments. (c) Analysis of the levels of V5-CARD11 protein in JURKAT NFκB-GFP reporter cell line infected with lentiviruses driving the expression of CARD11 wild type, CARD11 mutants S615F and E626K or empty vector. (d) NFκB-dependent GFP reporter activity in non-stimulated JURKAT cells expressing CARD11 wild type, CARD11 mutants S615F and E626K or empty vector. Bar graphs indicate the percentage of GFP positive cells analyzed by flow cytometry. Data is representative of 3 independent experiments. (e) NF-κB-dependent GFP reporter activity in JURKAT cells, after stimulation for 6h with 1 μg/ml ionomycin and increasing concentration of PMA (0.05, 0.5 and 5 nM), in JURKAT cells expressing CARD11 wild type, CARD11 mutants S615F and E626K or empty vector. Bar graphs indicate mean GFP intensity measured by flow cytometry across 3 biological replicates. (f) Western blot analysis of JNK phosphorylation in JURKAT cells infected with lentivirus driving the expression of V5-CARD11 wild type, V5-CARD11 mutants S615F and E626K or empty vector, after stimulation with 1 μg/ml Ionomycin and 25 nM PMA. The bar graphs in b, d and e show the mean values and error bars represent the s.d. P values were calculated using Student’s t test. WT, wild type

Article Snippet: We transfected the lentiviral constructs pLX304-blasV5 CARD11 wild type, CARD11 S615F, E626K, D230N, L215P, M183L and the empty vector with pCMV ΔR8.91 and pMD.G VSVG in HEK293T cells using Fugene 6 (Promega), according the manufacturers protocol.

Techniques: Functional Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Infection, Expressing, Flow Cytometry, Concentration Assay, Western Blot, Phospho-proteomics

Journal: Nature genetics

Article Title: The mutational landscape of cutaneous T-cell lymphoma and Sézary syndrome

doi: 10.1038/ng.3442

Figure Lengend Snippet: Functional characterization of Sezary syndrome cGKIβ mutations. (a) Schematic representation of the structure of the cGKIβ protein and sequence of the leucine zipper domain. Mutations are indicated with circles and mutated residues highlighted in red. (b,c) Western blot analysis (b) and quantification (c) of cGKIβ complex stability (urea dissociation) for wild type cGKIβ-HA/cGKIβ-V5, wild type cGKIβ-HA/cGKIβ-V5 E17K and wild type cGKIβ-HA/cGKIβ-V5 R21Q immunoprecipitates. (d) Western blot analysis of cGKIβ signaling (ERK activation and RhoA S188 phosphorylation) after 8-CPT-cGMP stimulation in JURKAT cells infected with virus driving the expression of wild type cGKIβ-V5, cGKIβ-V5 E17Q and cGKIβ-V5 R21Q mutants. (e) NFAT luciferase reporter assays in JURKAT cells expressing wild-type V5-cGKIβ, V5-cGKIβ E17K and V5-cGKIβ R21Q mutants 6 hours post stimulation with PMA (1μM) plus ionomycin (1 μg/ml). The bar graphs in (e) show the mean values and error bars represent the s.d. Data is representative of triplicate samples from two independent experiments. P values were calculated using two-tailed Student’s t test. WT, wild type

Article Snippet: We transfected the lentiviral constructs pLX304-blasV5 CARD11 wild type, CARD11 S615F, E626K, D230N, L215P, M183L and the empty vector with pCMV ΔR8.91 and pMD.G VSVG in HEK293T cells using Fugene 6 (Promega), according the manufacturers protocol.

Techniques: Functional Assay, Sequencing, Western Blot, Activation Assay, Phospho-proteomics, Infection, Virus, Expressing, Luciferase, Two Tailed Test