TEL-Syk is constitutively active and conferred hypersensitivity to low cytokine levels.

<p>(A) Autophosphorylation of TEL-Syk. HEK293T cells were transiently transfected with empty vector, Syk, TEL-Syk, and TEL-Syk KD. For the <i>in </i><i>vitro</i> kinase assay, cell lysates were immunoprecipitated with anti-Syk then incubated with P³² labeled ATP. Immunoblots demonstrating levels of Syk, TEL-Syk and TEL-Syk KD are shown below with Erk1/2 as a loading control. (B) Phosphorylation of NTAL and other cellular proteins by TEL-Syk. HEK293T cells were transiently co-transfected with NTAL plus empty vector, Syk, TEL-Syk, or TEL-Syk KD. Whole cell lysates were immunoblotted as indicated. Immunoblots are representative of three independent experiments. Note that expression of TEL-Syk was frequently lower than Syk or TEL-Syk KD in many experiments, likely because TEL-Syk is somewhat toxic to cells. Hence the TEL-Syk band is weaker in the whole cell lysate panel, despite the fact that overall tyrosine phosphorylation is much higher. (C-D) Expression of TEL-Syk leads to growth factor hypersensitivity in fetal liver hematopoietic progenitors. Syk-deficient fetal livers were transduced with retrovirus carrying empty vector, Syk, TEL-Syk, or TEL-Syk KD, sorted based on GFP expression then plated in methylcellulose at a range of cytokine concentrations. Total colony numbers (C), total cell numbers (D), and colony types (E) were determined 7 days after plating. (F) CFU-M brightfield images of vector and TEL-Syk at 0.1X cytokine stimulation were taken at 7 days after plating. Scale bars represent 100µm. Experiments were conducted in triplicate and data is expressed as ± SEM. Statistical significance was determined by one-way ANOVA. ***P< 0.001.</p

Expression of TEL-Syk led to increased STAT5 phosphorylation and promoted colony formation independent of JAK2 activity.

<p>(A) Lysates of sorted GFP⁺ and GFP^- fetal liver cells were separated by SDS-PAGE and immunoblotted with indicated antibodies. (B) Representative histograms of retrovirally transduced fetal liver cells cultured for 3 days in cytokines (IL-3, IL-6, SCF), starved for 6 hours, then restimulated with these cytokines. Cells were fixed, permeabilized and levels of phospho-STAT5 in the GFP⁺ cells was determined by flow cytometry. (C) Normalized MFI of phospho-STAT5 in retrovirally transduced fetal liver cultured for 3 days in cytokines, followed by a 6 hour starvation and 30 minute restimulation. Phospho-STAT5 MFIs were normalized to vector expressing cells. (D) Colony formation of sorted GFP+ fetal liver cells in methylcellulose in the presence of a JAK2 inhibitor. Colony numbers were normalized to a DMSO-treated (ie no inhibitor) control for each cell type and represented as a percent of maximal growth after 7 days in culture in 10 ng/mL GM-CSF. (E) Histograms of sorted GFP⁺ fetal liver cells starved for 6 hours and restimulated for 30 minutes with or without 50 ng/mL GM-CSF in the presence of the JAK inhibitor 1 at the indicated concentrations, stained for phospho-STAT5 and assessed by flow cytometry. Experiments were conducted in triplicate. Statistical significance was determined by one-way ANOVA. ***P< 0.001. Data is representative of 3 independent experiments.</p

TEL-Syk expression caused temporal increases in circulating inflammatory cytokines.

<p>(A) Pooled sera collected from TEL-Syk and vector expressing mice at 30, 45 and 60 days was examined using a Cytokine Profiler Array. Cytokine levels were represented as ratio of mean intensities (MI) of the given cytokine to a positive control after background intensity subtraction. (B) Representative histograms from 30-day old pooled sera collected from TEL-Syk and vector expressing mice using the Angiogenesis Array. Data are expressed as means ± SD. Statistical significance was determined by one-way ANOVA. **P< 0.01, ***P< 0.001.</p

TEL-Syk induced dyserythropoiesis <i>in vivo</i>.

<p>(A-D) Peripheral blood from BALB/c recipients injected with fetal liver cells transduced with the indicated retroviruses was collected at the indicated days after transfer and assessed by CBC analysis for (A) red blood cell counts, (B) hemoglobin levels, (C) mean corpuscular volume and (D) red cell distribution width. (E) Wright-Giemsa stains of peripheral blood smears from TEL-Syk and vector mice at 45 days following fetal liver transfer. Stomatocytes, dacrocytes, acanthocytes, and spheroctyes are indicated by *, **, ***, **** respectively. (F) Flow cytometry of peripheral blood from TEL-Syk and vector expressing mice. Peripheral blood was diluted in PBS and stained for anti-CD71 and anti-TER-119. Cells stained for lineage markers Gr-1, CD11b, TCRβ, CD19, B220, and F4/80 were excluded from analysis. (E) Quantitation of erythrocyte populations R1-R5 from panel F. Scale bars correspond to 100 µm. CBC data are shown as mean ± SEM for vector (n=11), Syk (n=11), TEL-Syk KD (n=11), TEL-Syk (n=11 at 30 days, n=9 at 45 days and n=7 at 60 days) and statistical significance was determined by one-way ANOVA. *P< 0.05, **P< 0.01.</p

TEL-Syk chimeric mice developed hypocellular splenomegaly with accelerated cellular apoptosis.

<p>(A) Spleens from vector, Syk, TEL-Syk and TEL-Syk KD expressing mice that were sacrificed at 60 days post cell transfer. (B) Splenic weights, (C) the ratio of splenocytes per mg of spleen, and (D) total numbers of splenocytes from chimeric mice. Data are shown as mean ± SEM for vector (n=14), Syk (n=12), TEL-Syk (n=10), TEL-Syk KD (n=14). (E) Flow cytometric analysis of splenic leukocytes. Data are shown as mean ± SEM for vector, Syk, TEL-Syk, TEL-Syk KD. (F, G) H&E stained sections of spleens from vector and TEL-Syk expressing mice. (H, I) Wright-Giemsa stained cytospins of splenocytes from vector or TEL-Syk chimeras. (J, K) Spleen sections from vector and TEL-Syk expressing mice stained with anti-cleaved capase-3 antibodies to indicate apoptotic cells. (L, M) Masson’s Trichrome stained sections of spleens from vector and TEL-Syk chimeras to indicate fibrosis. Scale bars correspond to 500 µm (F, G, J-M) and 10 µm (H, I). Statistical significance was determined by one-way ANOVA. **P< 0.01, ***P< 0.001.</p

TEL-Syk chimeric mice developed bone marrow hypocellularity and fibrosis.

<p>(A) Numbers of bone marrow cells isolated from mice receiving vector, Syk, TEL-Syk KD and TEL-Syk transduced fetal liver hematopoietic cells at 60 days following cell transfer. Data are shown as mean ± SEM for vector (n=14), Syk (n=13), TEL-Syk (n=7), TEL-Syk KD (n=14). Flow cytometric analysis of (B) leukocytes percentages and (C) total leukocyte numbers in the bone marrow cells. (D, E) H&E stained sternum sections from vector and TEL-Syk expressing mice, (F, G) Wright-Giemsa staining of bone marrow smears, and (H, I) cytospins of bone marrow cells isolated from femurs and tibias of TEL-Syk and vector expressing mice at 60 days following cell transfer. Dysplastic megakaryocytes (arrow) and neutrophils (asterisk) are indicated. (J, K) Reticulin-stained sternum sections from vector and TEL-Syk expressing mice. Scale bars correspond to 30 µm (D-G, J, K) and 10 µm (H, I). Data is shown as mean ± SEM and statistical significance was determined by one-way ANOVA. *P< 0.05, ***P< 0.001.</p

TEL-Syk expression reduced platelet numbers in the peripheral blood and megakaryocyte numbers in the bone marrow.

<p>(A) Platelet numbers were determined by CBC analysis, using a HEMAVET analyzer, of peripheral blood at the indicated days after fetal liver stem cell transfer. Due to high animal to animal variation (in part because of misinterpretation of red cell fragments by the analyzer), no statistical significance was observed (by one-way ANOVA) between groups. Data is shown as mean ± SEM for vector (n=11), Syk (n=11), TEL-Syk (n=11 at days 30 and 45, n=6 at day 60), TEL-Syk KD (n=11). Megakaryocyte numbers were quantitated per high power microscopic field in (B) bone marrow, (C) liver, and (D) spleen. An average of five fields were taken at 100X magnification of H&E sections. Data is shown as mean ± SEM for vector (n=5), Syk (n=5), TEL-Syk (n=5), TEL-Syk KD (n=5).</p

Additional file 1 of Pangenome analysis of Shewanella xiamenensis revealed important genetic traits concerning genetic diversity, pathogenicity and antibiotic resistance

Supplementary Material 1

Additional file 1 of Molecular characterization of extensively drug-resistant hypervirulent Pseudomonas aeruginosa isolates in China

Supplementary Material 1: Figure S1. Heatmap of XDR-hvPA carrying resistance genes; Figure S2. Heatmap of XDR-hvPA carrying virulence gene