Additional file 1 of GTSE1 promotes nasopharyngeal carcinoma proliferation and angiogenesis by upregulating STMN1

Supplementary Material

SDS-PAGE analysis proteolytic digestion of protoxins by <i>H</i>. <i>armigera</i> larval midgut juice.

(A) The digestion of Cry1Ac protoxin. (B) The digestion of Cry2Ab protoxin. M, the BlueRay prestained protein marker (MDBio, Qingdao, China). Both protoxins were incubated with midgut juice at different midgut juice protein/protoxin ratios (total protein content of the gut juice added/5 μg protoxin protein) for 1 h at 37°C.</p

Toxicity of Cry1Ac and Cry2Ab protoxins and their respective midgut juice-digested toxins against neonates of the susceptible SCD strain of <i>H</i>. <i>armigera</i>.

Toxicity of Cry1Ac and Cry2Ab protoxins and their respective midgut juice-digested toxins against neonates of the susceptible SCD strain of H. armigera.</p

The structure of Cry1Ac and Cry2Ab.

(A) The schematic diagram of Cry1Ac and Cry2Ab protoxins. The red dotted arrows represent cleavage sites. (B) Protein 3D structures of Cry1Ac protoxin and activated toxin. (C) Protein 3D structures of Cry2Ab protoxin and activated toxin. The three-dimensional structures of Cry1Ac and Cry2Ab protoxins were based on homologous modeling, built with SYBYL-Orchestrar software and viewed by the PyMOL program. The removed parts are shown in gray. The orange, green and purple represent domains I, II and III, respectively. Arg28 and Thr609 represent the cleavage sites of protease hydrolysis of Cry1Ac protoxin from the N- and C-termini, and Arg139 represents the cleavage sites of Cry2Ab from the N-terminus.</p

Magnitude and dominance of resistance to Cry1Ac in <i>H. armigera</i> associated with cadherin resistance alleles: <i>r</i>₁₅ in the cytoplasmic domain and <i>r</i>₁ in the extracellular region.

a<p>Concentration killing 50% of larvae and 95% fiducial limits (µg Cry1Ac per cm² diet).</p>b<p>Resistance ratio = LC₅₀ of a strain or F₁ progeny from a cross divided by LC₅₀ of the susceptible SCD strain.</p>c<p>Survival at the diagnostic concentration (1 µg Cry1Ac per cm² diet), n = 48.</p>d<p><i>h</i> ranges from 0 for completely susceptible to 1 for completely dominant.</p>e<p><i>h</i> calculated from LC₅₀ values <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053418#pone.0053418-Liu1" target="_blank">[34]</a>.</p>f<p><i>h</i> calculated from survival at the diagnostic concentration <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053418#pone.0053418-Liu1" target="_blank">[34]</a>.</p>g<p><i>h</i> is calculated only for F₁ progeny from crosses between resistant and susceptible strains.</p>h<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053418#s2" target="_blank">Results</a> pooled from the two reciprocal crosses.</p

Genetic linkage between the cytoplasmic domain mutant of <i>HaCad</i> (<i>r</i>₁₅) and resistance to Cry1Ac in the XJ-r15 strain of <i>H. armigera</i>.

<p>We crossed a female (<i>ss</i>) from the susceptible SCD strain with a male from the resistant XJ-r15 strain (<i>r</i>₁₅<i>r</i>₁₅) to produce the F₁ family (<i>r</i>₁₅<i>s</i>). Next we crossed an F₁ male (<i>r</i>₁₅<i>s</i>) with a susceptible SCD female (<i>ss</i>) to produce a backcross family from which larvae were placed on untreated diet (control) or diet treated with either 0.3 or 0.5 µg Cry1Ac per cm². After 5 days, all survivors were transferred to untreated diet, reared to the final instar, and genotyped. The frequency of heterozygotes (<i>r</i>₁₅<i>s</i>) relative to susceptible homozygotes (<i>ss</i>) was significantly higher for survivors on treated diet (68∶5) than for survivors on untreated diet (27∶23) (Fisher's exact test, P<0.0001).</p

Responses to Bt toxin Cry1Ac of <i>H. armigera</i> from a susceptible strain (SCD, blue), three resistant strains (red), and the F₁ progeny from crosses between each resistant strain and the susceptible strain (purple).

<p>SCD-r1: resistant strain with allele <i>r₁</i> affecting the extracellular domain of HaCad. XJ-r15 and AY-r15: resistant strains (from Xiajin and Anyang, respectively) with allele <i>r</i>₁₅ affecting the cytoplasmic domain of HaCad. Resistance ratio is the concentration killing 50% of larvae (LC₅₀) of each strain or group of F₁ progeny divided by the LC₅₀ for the susceptible SCD strain. The black bars show the 95% fiducial limits for LC₅₀.</p

Cadherin protein of <i>H. armigera</i> encoded by <i>HaCad</i>.

<p><b>A.</b> Protein structure of HaCad predicted from cDNA with extracellular region (amino-terminal signal sequence [SIG], 11 cadherin repeats <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053418#pone.0053418-Mendelsohn1" target="_blank">[1]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053418#pone.0053418-VanRensburg1" target="_blank">[11]</a>, membrane proximal region [MPR]), transmembrane region (TM), and cytoplasmic domain (C). <b>B.</b> Genomic DNA sequence of <i>HaCad</i>. Resistance allele <i>r₁</i> has a stop codon at 428G in cadherin repeat 3 caused by a genomic DNA deletion of ca. 10 kb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053418#pone.0053418-Yang2" target="_blank">[36]</a>. HaCad encoded by resistance allele <i>r</i>₁₅ lacks 55 amino acids in the cytoplasmic domain caused by a 165 bp deletion in exon 32. We found three genomic DNA variants of <i>r</i>₁₅ that cause loss of exon 32, one from each of three field populations: 1459 bp insertion from Xiajin, 92 bp deletion from Anyang, and >5000 bp insertion from Anci.</p

Cry1Ac binding to Sf9 cells transfected with four alleles of <i>HaCad</i>.

<p><i>s</i>: susceptible allele. <i>r</i>₁₅: resistant allele, encoding cadherin with a 55 amino acid deletion in the cytoplasmic domain (C). <i>s</i>/<i>r</i>₁₅: chimeric allele with C from <i>r</i>₁₅ and the other components from <i>s</i>. <i>r</i>₁₅/<i>s</i>: complementary chimeric allele with C from <i>s</i> and the other components from <i>r</i>₁₅. Cells were treated with 10 nM Cry1Ac, then probed sequentially with anti-Cry1Ac antiserum (1∶100) and FITC-conjugated anti-rabbit antibody (1∶100). No Cry1Ac binding was detected in control cells that were either transfected with an empty bacmid (EB) or not transfected (Sf9).</p

Non-Recessive Bt Toxin Resistance Conferred by an Intracellular Cadherin Mutation in Field-Selected Populations of Cotton Bollworm

<div><p>Transgenic crops producing <em>Bacillus thuringiensis</em> (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, <em>Helicoverpa armigera</em>. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.</p> </div