Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: A transient ternary “Molecular Sandwich” type complex (MSC) is a part of the PAI-1 inhibitory mechanism in vivo.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: In Vivo

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: Crystal structures of PAI-1 (yellow) complexes with S195A tcuPA (blue) (33), and SMB domain of Vn (brown) (34) were used. The exposed RCL of active PAI-1 is shown in red with positions of E350 and E351 (P4′P5′ nomenclature of Schechter and Berger (53)) in blue, β-sheet A in green and a-helix F in cyan. Active site A195 of S195A tcuPA is shown as a white space-filled residue, and positions of positively charged residues of 37-loop of uPA are red.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques:

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: Scheme 3: A transition intermediate (I‡), which is in equilibrium with the active PAI-1 (IA), forms during conformational changes accompanying the transition to the latent conformation (IL) (46). A diagram depicts how IA is stabilized in the complex with ligand(s) (IA1) due to an increase in the ΔG‡ (ΔΔG‡ = ΔG‡1 - ΔG‡0), where ΔG‡1 and ΔG‡0 are changes in the activation Gibbs free energy for active PAI-1 with and without ligand(s), respectively. A bar graph shows the contribution of Vn (black), S195A tcuPA (white), and both ligands (gray) to ΔΔG‡ for rPAI-1, Q123K PAI-1, and Gl-PAI-1 at 37°C. The values of ΔG‡0 and ΔG‡1 were calculated as ΔG‡ = ΔH‡ - TΔS‡ (Table 3).

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: Activation Assay

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: Linear dependences of kobs from [ligand] for the association of PAI-1 and its complexes with ligands (k+n; n=1–4; Scheme 1). Values of k+n for the interaction of S195A tcuPA with NBD P1′ PAI-1 (○); Vn and NBD S119C PAI-1 (□); S195A tcuPA with NBD P1′ PAI-1/Vn (●), and Vn with NBD S119C PAI-1/S195A tcuPA (■) were calculated from the slopes of the best linear fit to the data (Table 1). Slow dissociation of S195A tcuPA limits the reaction between tPA and complex of NBD P9 PAI-1 with S195A tcuPA (△) and MSC (complex of NBD P9 PAI-1 with S195A tcuPA and Vn; Scheme 1) (▲).

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques:

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: The values of the association and dissociation rate constants (k +1 , k +2 , k +3 , k +4 and k −1 , k −2 , k −3 , k −4 respectively); and dissociation equilibrium constants (K D = k − /k + ) for the interactions between wt PAI-1, its mutant variants, and their complexes with S195A tcuPA and Vn at 25°C.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: Mutagenesis, Variant Assay

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: The dependences of kobs for the fast step (an increase in NBD fluorescence emission) (○), and slow step (a decrease in the NBD fluorescence emission) (●) on enzyme concentration. The solid lines represent the best fit of the linear equation kobs2 = (klim+k−1) + k1[tcuPA] (r2=0.93) (fast step), and a hyperbolic equation kobs1 = klim*[tcuPA]/(Km + [tcuPA]) (r2=0.99) (slow step) to the data by SigmaPlot 11.0. The values of klim and Km (Scheme 2) were 33.3 ± 5.2 s−1, and 10.2 ± 2.2 μM, respectively. The slope and intercept of the linear dependence of kobs on enzyme concentration represent a second order rate constant for the interaction of tcuPA with NBD P1′ PAI-1 k+1 = 26.8 ± 2.8 μM−1 s−1, and (klim+k−1) = 50 ± 8 s−1, respectively. Inset: Trace of the changes in NBD fluorescence for the reaction of NBD P1′ PAI-1 (0.12 μM) with 2.0 μM tcuPA. Values of kobs1 and kobs2 were calculated by fitting (grey line) a double exponential equation to the time traces of changes in NBD-fluorescence emission using SigmaPlot 11.0.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: Fluorescence, Concentration Assay

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: Two step inactivation of proteianase (E) by PAI-1 (I)

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques:

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: (A) SDS PAGE analysis of products of the reaction between sctPA and rPAI-1, Q123K PAI-1, and Gl-PAI-1 and their complexes with Vn, S195A tcuPA, and both ligands (the table at the top of the gels.) incubated for 0, 1, 15, 90 (stained with SYRRO Ruby) and 720 h (PAI-1 antigen was visualized with Western blot analysis) at 37°C. Positions of PAI-1/tPA SIC (1), Vn (2), tPA (3), S195A tcuPA (4), latent and cleaved rPAI-1 (5 and 6, respectively), co-migrating S195A tcuPA and latent Gl-PAI-1 (7), cleaved Gl-PAI-1 (8), are indicated to the left (rPAI-1 and Q123K PAI-1) and to the right (Gl-PAI-1) of the gels. (B) Active PAI-1 concentration in the reaction mixtures, shown in the Western blot (A, lower panel) was estimated by inhibiting the activation of Plg by uPA, as described elsewhere (9;41). After incubation with both Vn and S195A tcuPA for 1 month (720h) at 37°C rPAI-1 (filled bars) and Gl-PAI-1 (gray bars), but not Q123K PAI-1 (empty bars) inhibit the activation of Plg by uPA.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: SDS Page, Incubation, Staining, Western Blot, Concentration Assay, Activation Assay

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: The concentration of active PAI-1 was determined from the residual tPA amidolytic activity after titrating the sample with increasing amounts of sctPA as described under Experimental Procedures and plotted on a semilogarithmic scale (filled symbols). Separate aliquots were incubated with an excess of sctPA and the products of the reaction were separated by SDS PAGE, as shown in Figure 4 and described under Experimental Procedures. The amounts of active PAI-1 were estimated from relative density of SIC, latent and cleaved PAI-1 on gels and plotted as empty symbols. Linear equations were fit to both sets of data (solid lines represent the best fits to filled symbols) using SigmaPlot 11.0, and the first order rate constants (kL1, kL2, kL3, kL4 (Table 2), and kL1SDS PAGE, kL2SDS PAGE, kL3SDS PAGE, kL4SDS PAGE respectively, Scheme 1) were calculated from the slopes. Inset: Correlation between values of kL measured by two different methods. The values of kL estimated from the results of SDS PAGE analysis were plotted against the corresponding values obtained from titrating the reaction mixtures with sctPA: rPAI-1 (empty symbols), Gl-PAI-1 (black symbols), and Q123K PAI-1 (grey symbols). Data for free rPAI-1, Gl-PAI-1, and Q123K PAI-1 are shown as circles, for their complexes with Vn as triangles, with S195A tcuPA as reversed triangles, and for both ligands as squares. The solid line represents the best linear fit to the data of linear equation (kLSDS PAGE=−0.27+ 0.93 kL; r2= 0.99).

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: Concentration Assay, Activity Assay, Incubation, SDS Page

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: Values of k L1 , k L2 , k L3 and k L4 ( Scheme 1 ) for rPAI-1, Q123K PAI-1, Gl-PAI-1 and their complexes with Vn, S195A tcuPA and both ligands, determined at different temperatures.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques:

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: A linear correlation between changes in the contribution of -TΔS‡ and ΔH‡ (Table 3) to ΔG‡ for the three PAI-1 variants and their complexes with ligands at 37°C. The values of -TΔS‡ for rPAI-1 (empty symbols), Gl-PAI-1 (black symbols), and Q123K PAI-1 (grey symbols) for free serpins (circles), and their complexes with Vn (triangles), S195A tcuPA (reversed triangles), and both ligands (squares) were plotted as a function of ΔH‡. The solid line represents the best fit of the linear equation (-TΔS‡ =90.6-0.94 ΔH‡; r2=0.99) to the data.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques:

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: MSCs formed with S195A tcuPA and mAbs protect rPAI-1/Vn from inactivation by MA-33B8 and spontaneous active to latent transition. Dependences of kobs for RCL insertion for NBD P9 PAI-1 (○), NBD P9 PAI-1/Vn (●) and MSCs with MA-56A7C10 (△), MA-44E4 (□), and S195A tcuPA (▽) on [MA-33B8]. NBD P9 PAI-1, its complex with Vn (10 nM), and MSCs formed in presence of 20 nM of S195A tcuPA or a mAb (MA-56A7C10 or MA-44E4) were incubated with MA-33B8 (50–400 nN) in 96-well flat bottom plates from Costar (Corning Inc) and an increase in NBD fluorescence with time was registered using a fluorescence spectrophotometer SynergyTM HT Hybrid Reader. The kobs were calculated by fitting a single exponential equation to the data using Gen5™ 2.0 Data Analysis Software (BioTek) and SigmaPlot 11.0 (SPSS Inc.), as described elsewhere (26;43;45). Inset: Stabilization of rPAI-1/Vn (lane A) in MSCs with S195A tcuPA (lane B), MA-56A7C10 (lane C), and MA-42A2F6 (lane D) after incubation at 37°C for 168 h (one week). Active PAI-1 was quenched by sctPA and reaction mixtures were analyzed as described under Experimental Procedures. Positions of mAb (1), PAI-1/tPA SIC (2), Vn (3), tPA (4), S195A tcuPA (5), latent (6) and cleaved PAI-1 (7) are indicated to the right of the gel.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: Incubation, Fluorescence, Spectrophotometry, Software

Journal: Neuropsychopharmacology

Article Title: Identification of MicroRNA-124-3p as a Putative Epigenetic Signature of Major Depressive Disorder

doi: 10.1038/npp.2016.175

Figure Lengend Snippet: Functional validation of miR-124-3p targets. (a) Bar diagram represents the relative quantification of GRIA4, NR3C1, HSP90AB1, and AKT1S1 transcripts in SH-SY5Y neuroblastoma cell line transiently transfected with non-target-specific scramble control (n=3), mimic miR-124-3p overexpression oligo (n=3), anti-miR-124-3p overexpression oligo (n=3), and vehicle control (n=3). The expression status of respective genes was quantified in treatment groups relative to vehicle control using one-way analysis of variance, followed by post hoc Tukey's multiple comparison test. GAPDH was used as normalizer for target gene expression. Values are represented as ±SEM ‘a' and ‘b' denote significant differences when compared with vehicle. Overall group differences in the four groups (vehicle, scramble, mimic, and anti) are as follows: GRIA4: df=3.8; f=2.95, p=0.09; NR3C1: df=3.8; f=32.15, p<0.001; HSP90AB1: df=3.8; f=4.92, p=0.03; AKT1S1: df=3.8; f=6.61, p=0.015. ap<0.029; bp<0.001. (b) Schematic representation of Gria4 3′ untranslated region (UTR) cloning strategy with restriction enzyme digestion (SpeI and HindIII restriction enzyme digestion sites) in pMIR-Report vector downstream of firefly luciferase reporter gene. (c) Relative luciferase activity (normalized with Renilla luciferase activity) driven by Gria4 3′ UTR via miR-124-3p interaction was determined in HEK-293 lysate co-transfected with reporter clone and individually mimic miR-124-3p oligo (n=3) or antisense (anti) miR-124-3p oligo (n=3). Representative data were compared with nonspecific scramble control (n=3) and expressed as ±SEM. Overall group differences in the three groups (scramble, mimic, and anti) are as follows: df=2.6; f=33.22, p=0.001. ap=0.023; bp=0.011. (d–h) Involvement of endogenous miR-124-3p binding on single 7mer-m8 site of Gria4, two different sites of Nr3c1, one 8mer site of Akt1s1, one 8mer site of Gria3, and the proximal 7mer-A1 site of Grin2a on their 3′ UTR determined by RNA-induced silencing complex (RISC)-mediated immunoenrichment assay. Relative 3′ UTR enrichment normalized to 10% input in the immunoprecipitated ribonucleoprotein complex was analyzed with qPCR. Data are represented as ±SEM. Data were analyzed by independent-sample ‘t' test. ‘a' denotes significant difference between vehicle and corticosterone (CORT)-treated groups. Gria4 p=0.022, Nr3c1 7mer-m8 p=0.034, Nr3c1 7mer-A1 p=0.133, Akt1s1 p=0.58, Gria3 p=0.16 and Grin2a p=0.39.

Article Snippet: Amplified 3′ UTR was double-digested ( Spe I and Hind III) and directionally cloned in pMIR luciferase reporter vector (Addgene, Cambridge, MA, USA) ( Supplementary Table S1 ).

Techniques: Functional Assay, Transfection, Over Expression, Expressing, Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Binding Assay, Immunoprecipitation

Journal: Oncotarget

Article Title: Genetic polymorphism of SLC31A1 is associated with clinical outcomes of platinum-based chemotherapy in non-small-cell lung cancer patients through modulating microRNA-mediated regulation

doi: 10.18632/oncotarget.24794

Figure Lengend Snippet: ( A ) Schematic structure of SLC31A1 gene and linkage disequilibrium of its candidate SNPs (color intensity is proportional to D ′ and percent numbers represent r 2 ). ( B ) Local sequence of SLC31A1 3′UTR annotated with the close locations of rs10759637 and two putative binding sites for hsa-miR-29 family. ( C ) Comparison of secondary structures, built with RNAfold program, of the local SLC31A1 3′UTRs harboring hsa-miR-29 binding sites and the wild or the variant alleles of rs10759637. ( D ) Luciferase reporter assays in 16HBE cells to analyze the regulatory role hsa-miR-29 on SLC31A1 3′UTR that could be modulated by rs10759637. Co-transfection with hsa-miR-29 member (a, b, or c) and SLC31A1 3′UTR construct consistently resulted in reduced luciferase activity. In the cases of hsa-miR-29 b or c, rs10759637 allelic state significantly affected the cis -regulation toward reporter expression. ( E ) Real-time quantitative RT-PCR analysis of SLC31A1 mRNA expression in lung tumorous tissues from an independent cohort of patients, which was significantly correlated with rs10759637 genotypes.

Article Snippet: Human bronchial epithelial cell line 16HBE (1.0 × 10 5 cells) was cotransfected with wild or variant rs10759637 report vector (400 ng) and microRNA (40pmol miRNA) using Lipofectamine ™ 2000 (Invitrogen).

Techniques: Sequencing, Binding Assay, Variant Assay, Luciferase, Cotransfection, Construct, Activity Assay, Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Genetic polymorphism of SLC31A1 is associated with clinical outcomes of platinum-based chemotherapy in non-small-cell lung cancer patients through modulating microRNA-mediated regulation

doi: 10.18632/oncotarget.24794

Figure Lengend Snippet: Association between SLC31A1 SNPs and toxicity outcomes

Article Snippet: Human bronchial epithelial cell line 16HBE (1.0 × 10 5 cells) was cotransfected with wild or variant rs10759637 report vector (400 ng) and microRNA (40pmol miRNA) using Lipofectamine ™ 2000 (Invitrogen).

Techniques:

Journal: Oncotarget

Article Title: Genetic polymorphism of SLC31A1 is associated with clinical outcomes of platinum-based chemotherapy in non-small-cell lung cancer patients through modulating microRNA-mediated regulation

doi: 10.18632/oncotarget.24794

Figure Lengend Snippet: Stratification analysis of association between SLC31A1 SNPs and toxicity outcomes

Article Snippet: Human bronchial epithelial cell line 16HBE (1.0 × 10 5 cells) was cotransfected with wild or variant rs10759637 report vector (400 ng) and microRNA (40pmol miRNA) using Lipofectamine ™ 2000 (Invitrogen).

Techniques:

Journal: Oncotarget

Article Title: Genetic polymorphism of SLC31A1 is associated with clinical outcomes of platinum-based chemotherapy in non-small-cell lung cancer patients through modulating microRNA-mediated regulation

doi: 10.18632/oncotarget.24794

Figure Lengend Snippet: Association between SLC31A1 SNPs and survival

Article Snippet: Human bronchial epithelial cell line 16HBE (1.0 × 10 5 cells) was cotransfected with wild or variant rs10759637 report vector (400 ng) and microRNA (40pmol miRNA) using Lipofectamine ™ 2000 (Invitrogen).

Techniques:

Journal: Oncotarget

Article Title: Genetic polymorphism of SLC31A1 is associated with clinical outcomes of platinum-based chemotherapy in non-small-cell lung cancer patients through modulating microRNA-mediated regulation

doi: 10.18632/oncotarget.24794

Figure Lengend Snippet: Stratification analysis of association between SLC31A1 SNPs and overall survival

Article Snippet: Human bronchial epithelial cell line 16HBE (1.0 × 10 5 cells) was cotransfected with wild or variant rs10759637 report vector (400 ng) and microRNA (40pmol miRNA) using Lipofectamine ™ 2000 (Invitrogen).

Techniques: Variant Assay

Journal: Oncotarget

Article Title: Genetic polymorphism of SLC31A1 is associated with clinical outcomes of platinum-based chemotherapy in non-small-cell lung cancer patients through modulating microRNA-mediated regulation

doi: 10.18632/oncotarget.24794

Figure Lengend Snippet: In the entire patients ( A ) and subgroups stratified respectively with gender ( B ), age ( C ), smoking status ( D ), ECOG performance status ( E ), and histological type ( F ), the curves were plotted with SPSS software according to genotypes of two SNPs. For rs2233914, the A/A homozygote was compared to the G/G+G/A group as reference. For rs10759637, the A/C heterozygote was compared to the A/A+C/C homozygotes group as reference.

Article Snippet: Human bronchial epithelial cell line 16HBE (1.0 × 10 5 cells) was cotransfected with wild or variant rs10759637 report vector (400 ng) and microRNA (40pmol miRNA) using Lipofectamine ™ 2000 (Invitrogen).

Techniques: Software

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) CW EPR spectra of TACC3 MTSL-C828 (black) and TACC3 MTSL-C662 (red) at 120 K. ( B-C ) Normalized four-pulse DEER trace at 50 K for (B) TACC3 MTSL-C828 and (C) TACC3 MTSL-C662. Inset, traces after subtraction of a mono-exponential decay. The weak oscillation is most likely from residual proton modulation.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques:

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) In vitro co-precipitation assays between immobilized NusA-TACC3 TACC domain and ch-TOG truncates. ( B ) Superposition of the top 20 ch-TOG 1817-1957 NMR structures using the structured core of helices, H1-H4 (residues 1826-1894) for alignment. Inset, structures aligned on ch-TOG residues 1905-1915. ( C ) Cartoon representation of the best NMR structure for ch-TOG 1817-1957 shown in the same orientation as in (B). Inset, stick representation of ch-TOG residues 1905-1915. Structures in (B-C) are colored by spectrum mode where the N-terminus is blue and the C-terminus is red.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: In Vitro

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) Chemical shift perturbations observed on interaction of 2 H/ 15 N labeled ch-TOG 1817-1957 with a three-fold excess of TACC3 629-838 Δ699-765. Data extracted from the spectra shown in . ( B ) MST binding experiment of a synthetic ch-TOG H5 peptide with TACC3 629-838 Δ699-765. ( C ) Cartoon representation of an AlphaFold2 Multimer model of the complex between the TACC3 629-838 Δ699-765 dimer (wheat) and ch-TOG 1817-1957 (green). Arrows indicate the position of the deletion in TACC3. The model is shown below colored according to per residue confidence score (pLDDT) in rainbow colors from high (blue) to low (red) confidence. ( D ) Cartoon representations of the AlphaFold2 model showing the interface between the H5 region of ch-TOG (green) and TACC3 (wheat). Sidechains contributing to the interface from TACC3 protomer A (orange), TACC3 protomer B (yellow) and ch-TOG H5 (green), are shown in stick representation.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: Labeling, Binding Assay, Residue

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: Plots show the protein alone (blue) and in the presence of a three-fold excess of TACC3 629-838 Δ699-765 (red).

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques:

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) Predicted aligned error (PAE) plot for a model of the complex between TACC3 629-838 Δ699-765 and ch-TOG 1817-1957 generated by AlphaFold2 Multimer. ( B ) In vitro co-precipitation assays between immobilized His-NusA-ch-TOG 1817-1957 constructs and TACC3 629-838 (top). Binding of TACC3 was resolved by Western blot (bottom). ( C ) Sequence coverage map of TACC3 629-838 Δ699-765 in HDX-MS experiments. The yellow shaded regions in the thick bar at the top of the panel represent regions with sequence coverage while gray indicates regions that were not covered by detected peptides. Narrow yellow bars represent the individual peptides detected. ( D-F ) Woods plots showing the differences in deuterium uptake in TACC3 at three HDX timepoints (0.5, 5, 30 min of HDX), comparing TACC3 629-838 Δ699-765 alone with TACC3 629-838 Δ699-765 in the presence of Affimers E4 (D), E7 (E) and E8 (F). Woods plots were generated using Deuteros 2.0. Peptides colored in blue are protected from hydrogen/deuterium exchange in the presence of Affimers. Peptides with no significant difference in exchange between conditions, determined using a 99 % confidence interval and a hybrid statistical test (dotted line), are shown in gray. A summary of key details of the HDX-MS experiment is shown in .

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: Generated, In Vitro, Construct, Binding Assay, Western Blot, Sequencing

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) In vitro co-precipitation assay between Affimers, TACC3 and ch-TOG. C-terminal His-tagged Affimers were immobilized on nickel sepharose resin and incubated with TACC3 629-838 (TACC3 TD) or TACC3 629-838 Δ699-765 (TACC3 TDΔ). Binding of ch-TOG 1517-1957 in the presence of Affimer was assessed by the addition of ch-TOG to TACC3 TDΔ reactions. ( B ) ELISAs to assess binding between Affimers and TACC3 629-838 Δ699-765. Biotinylated TACC3 629-838 Δ699-765 was immobilized on Streptavidin coated plates and incubated with an Affimer dilution series (orange circles). Background binding of Affimers to the plate was measured by incubating the proteins in wells coated with PBS (gray squares). Data points are the mean ± standard error of the mean from two experiments. ( C ) Woods plots describing differences in deuterium uptake by residue, after 30 minutes of exchange, between TACC3 629-838 Δ699-765 in the absence of binding partner and in the presence of Affimers (as indicated). Woods plots were generated using Deuteros 2.0. Peptides colored in blue are protected from hydrogen/deuterium exchange in the presence of Affimers. Peptides exhibiting no significant difference in exchange between conditions, determined using a 99% confidence interval and a hybrid statistical test (dotted line), are shown in gray. ( D ) Cartoon representation the TACC3 629-838 Δ699-765–ch-TOG complex model with TACC3 colored according to HDX behavior as in (C). The region of TACC3 protected from hydrogen/deuterium exchange in the presence of Affimers E4, E7 and E8 is colored blue, and ch-TOG H5 is colored pink. ( E ) Pull-down assay using cell extracts from asynchronously growing HeLa cells expressing GFP-TACC3 and the indicated mCherry-Affimers, or mCherry as a control. GFP-TACC3 was immunoprecipitated using GFP-trap and a representative Western blot from three experiments is shown. Blots were probed for TACC3, mCherry and ch-TOG, as indicated. ( F ) Quantification of mCherry and ch-TOG bands from pull-down assays. Each dot represents the mean intensity of the indicated protein band normalized to the mCherry condition, colored by experiment. Crossbar indicates the mean from three experiments.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: In Vitro, Incubation, Binding Assay, Residue, Generated, Pull Down Assay, Expressing, Control, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) Representative confocal micrographs of metaphase HeLa cells (left) and CRISPR GFP-FKBP-TACC3 HeLa cells (right) expressing the indicated mCherry-Affimers (red). Cells were fixed with PTEMF or ice-cold methanol, as indicated. Where parental HeLa cells were used, an antibody to stain endogenous TACC3 (green) was used. ( B ) Induced relocalization of TACC3 Affimers to mitochondria. Representative confocal micrographs of HeLa cells at metaphase expressing the indicated FKBP-GFP-Affimers (green) with dark-MitoTrap, that were either treated or not with rapamycin (200 nM, 30 min) prior to fixation. Cells were stained for tubulin (not shown in merge) and TACC3 (red). DNA (blue) is shown in the merge. Relocalization of the Affimer to mitochondria can be seen in the rapamycin-treated cells compared to control, but no relocation of TACC3 is observed. ( C ) Quantification of Affimer (x-axis) and TACC3 (y-axis) spindle localization in untreated cells (salmon) and rapamycin treated cells (turquoise). Spindle localization was calculated as the ratio of spindle to cytoplasmic fluorescence shown on a log2 scale, n = 11-22 cells per condition. ( D ) Widefield micrographs of live HeLa cells in metaphase expressing GFP-TACC3 (green) and TACC3 Affimers (red) labeled with mCherry at either the C or N-terminus as indicated. Scale bars, 10 µm.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: CRISPR, Expressing, Staining, Control, Fluorescence, Labeling

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) Representative confocal micrographs of untreated or MLN8237-treated (0.3 µM, 40 min) knock-in GFP-FKBP-TACC3 HeLa cells in metaphase. Cells were stained for ch-TOG (red), DNA (blue) and GFP-boost antibody was used to enhance the signal of GFP-FKBP-TACC3 (green). ( B ) Cells expressing mCherry or mCherry-Affimers, as labeled. Scale bars, 10 µm. ( C ) Quantification of spindle recruitment of TACC3 (green) and ch-TOG (red). Each dot represents a single cell, n = 40-45 cells per condition pooled from three independent experiments. Dashed line, no spindle enrichment. Large dot and bars, mean ± one standard deviation. Analysis of variance (ANOVA) followed by Tukey’s post hoc test is shown above each group, using the untransfected and untreated cells (black) and untransfected MLN8237-treated cells (purple) for comparison. ***, p < 0.001; NS, > 0.05.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: Knock-In, Staining, Expressing, Labeling, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1101/2024.05.31.596836

Figure Lengend Snippet: ( A ) Representative confocal micrographs of untreated or MLN8237-treated (0.3 µM, 40 min) knock-in CLTA-FKBP-GFP HeLa cells at metaphase. Cells were fixed in PTEMF and stained for TACC3 (red), DNA (blue), and a GFP-boost antibody was used to enhance the signal of CLTA-FKBP-GFP (green). ( B ) Cells expressing the indicated mCherry-Affimers (grey, not shown in merge). Scale bar, 10 µm.

Article Snippet: The following plasmids were available from previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629-838, pETM6T1 TACC3 629-838 Δ699-765, pETM6T1 ch-TOG 1517-1957 and truncated ch-TOG constructs in pETM6T1 ( ; ); mEmerald-γ-tubulin was from Addgene #54105.

Techniques: Knock-In, Staining, Expressing