7 research outputs found
Additional file 2: of Novel proteome and acetylome of Bemisia tabaci Q in response to Cardinium infection
Table S1. List of proteins identified and quantified by the TMT analysis in the experiment. Table S2. List of differential expressed proteins by the TMT analysis in the experiment. Table S3. Subcellular location of differentially expressed proteins compared C*+ to C− strains. Table S4. Gene Ontology classification of differentially expressed proteins compared C*+ to C− strains. Table S5. GO annotation of differentially expressed proteins compared C*+ to C− strains. Table S6. KEGG pathway of differentially expressed proteins compared C*+ to C− strains. Table S7. Detailed information on identified acetylated peptides compared C*+ to C− strains. Table S8. Detailed information of differentially expressed acetylation peptides compared C*+ to C− strains. Table S9. Amino acid sequence analysis from the − 10 to + 10 positions around the acetylated lysine. Table S10. GO functional classification of differentially expressed acetylated proteins compared C*+ to C− strains. Table S11. GO enrichment analysis of differentially expressed lysine acetylated proteins using Blast2GO. Table S12. KEGG pathway enrichment analysis of differentially expressed acetylated proteins compared C*+ to C− strains. Table S13. Domain enrichment analysis of differentially expressed acetylated proteins compared C*+ to C− strains. Table S14. Interaction networks of differentially expressed Kac proteins compared C*+ to C− strains. (ZIP 953 kb
Additional file 1: of Novel proteome and acetylome of Bemisia tabaci Q in response to Cardinium infection
Figure S1. Amplification product of Cardinium-infected Bemisia tabaci Q using specific primers. 1–10: Cardinium-infected B. tabaci Q; +: positive control; −: negative control; M: DNA marker. Figure S2. The subcellular location of up- (A) and down-(B) regulated proteins compared C*+ to C− strains. Figure S3. GO distribution of up-regulated proteins in biological process (A), cellular component (B) and molecular function terms (C) compared C*+ to C− strains. Figure S4. GO classification of down-regulated proteins in biological process (A), cellular component (B) and molecular function terms (C) compared C*+ to C− strains. (ZIP 158 kb
Naïve CD8<sup>+</sup> but not CD4<sup>+</sup> T cells were significantly decreased in the periphery of Tsc1 KO mice.
<p>The peripheral T cell subsets of WT and Tsc1 KO mice were detected using FCM 4–5 wks after birth. <b>A.</b> Total thymic cell number and thymocyte subset profile of Tsc1 KO mice were identical as WT littermates. <b>B.</b> Total cell number of spleen, pLNs and mLNs of WT and Tsc1 KO mice. <b>C.</b> The percentage of CD8<sup>+</sup>T but not CD4<sup>+</sup>T cells decreased dramatically in all peripheral lymphoid organs of Tsc1 KO mice. <b>D.</b> The FACS profile analysis of CD62L, CD45RB and CD44 on pLN CD4<sup>+</sup> T cells was evaluated. The frequency of naïve CD62L<sup>hi</sup>, CD45RB<sup>hi</sup> and CD44<sup>low</sup> CD4<sup>+</sup>T cells in spleen, pLN and mLN of Tsc1 KO mice was summarized. <b>E.</b> The FACS profile analysis of CD62L, CD45RB and CD44 on the gated pLN CD8<sup>+</sup> T cells was evaluated. The frequency of naïve CD62L<sup>hi</sup>, CD45RB<sup>hi</sup> and CD44<sup>low</sup> CD8<sup>+</sup>T cells in spleen, pLN and mLN of Tsc1 KO mice was summarized. *p<0.05; **p<0.01; ***p<0.001. Data were shown as Mean±SD (N = 6).</p
Decreased Akt-FoxO1/FoxO3a phosphorylation of Tsc1 KO naïve CD8<sup>+</sup>T cells in response to IL-7.
<p>The expression of CD122, CD127 and CD95 on naïve CD8<sup>+</sup>T cells was determined by gating CD8<sup>+</sup>CD44<sup>low</sup> cells. Comparable surface CD122, CD127 (<b>A</b>) and CD95 (<b>B</b>) expression in Tsc1 KO naïve CD8<sup>+</sup> T cells. The dash-dot line represents staining with an isotype control antibody. The histograms (gray) represent WT whereas the open histograms with solid line show Tsc1 KO staining pattern. Cells are gated with CD8<sup>+</sup>CD44<sup>low</sup> population. Representative data are shown from one of two separate experiments, with three mice in each group. (<b>C</b>) Western blot analysis of mTORC2-Akt-FoxO1/FoxO3a-Bim axis in naïve CD8<sup>+</sup> T cells after stimulation with IL-7. Sorted naïve CD8<sup>+</sup>T cells were cultured with IL-7 in the presence of Rapa or not for 24 hrs. The freshly isolated WT or Tsc1 KO naïve CD8<sup>+</sup>T cells were used as a control. One representative is shown from two or three separate experiments.</p
TSC1 regulates naïve CD8<sup>+</sup> T cell survival to IL-7 or IL-15 in a rapamycin-insensitive manner.
<p>Sorted WT and Tsc1 KO mouse naïve CD8<sup>+</sup>CD44<sup>low</sup> T cells were cultured in medium alone or supplemented with IL-15 or IL-7(in the presence or absence of Rapa) for 24 hrs or the below indicated time points. (<b>A</b>) Significantly decreased live cell number of Tsc1 KO naïve CD8<sup>+</sup>T cells than WT naïve CD8<sup>+</sup>T cells in the presence of IL-15 or IL-7 for 24 hours. Live cells were determined by trypan blue exclusion assay. (<b>B</b>) Tsc1 KO naïve CD8<sup>+</sup> T cells showed severe atrophy in the presence of IL-7 for 24 hrs. (<b>C</b>) One representative of PI staining in gated CD8<sup>+</sup>T cells after culture with or without IL-7 and IL-15. (<b>D</b>) Percentage of cell death was measured by PI staining and statistically analyzed. The above data were one representative of three separate experiments, with three wells in each group. (<b>E</b>) Representative staining of CD45.2 and CD8 for pLN cells of CD45.1<sup>+</sup> host in the presence or absence of Rapa 7 days after transfer. Either sorted WT or Tsc1 KO CD45.2<sup>+</sup> naïve CD8<sup>+</sup> T cells were adoptively transferred into sublethally irradiated(4 Gy) CD45.1<sup>+</sup> host and the donor cells were determined by CD8 and CD45.2 staining. (<b>F</b>) Significantly decreased percentage and cell number of Tsc1 KO CD45.2<sup>+</sup> naïve CD8<sup>+</sup> T cells in spleen of sublethally irradiated (4 Gy) CD45.1<sup>+</sup> host in the presence or absence of Rapa. (<b>G</b>) Significantly decreased percentage and cell number of Tsc1 KO CD45.2<sup>+</sup> naïve CD8<sup>+</sup> T cells in pLNs of sublethally irradiated (4 Gy) CD45.1<sup>+</sup> host in the presence or absence of Rapa. Data were shown as Mean±SD (3 mice each group). *p<0.05; **p<0.01; ***p<0.001 compared with the indicated groups.</p
Generation of the T cell-specific Tsc1 KO mice.
<p>(<b>A</b>) Western blot analysis showed TSC1 was expressed in thymus, spleen as well as pLNs of WT B6 mice, brain serves as the positive control. (<b>B</b>) Tsc1 and Tsc2 expression level in splenic CD8<sup>+</sup>T cells was higher than that in B cells and CD4<sup>+</sup>T cells as detected by Real-time PCR analysis. *p<0.05; **p<0.01; ***p<0.001 compared with the indicated groups. Date were shown as Mean±SD (N = 6). (<b>C</b>) Schematic representation of deletion of Tsc1 exons 17 and 18 by Lck-Cre-mediated recombination in T cells. (<b>D</b>) Genotyping of wild type (Tsc1<sup>+/+</sup>, WT), heterozygous(Lck-cre<sup>+</sup>Tsc1<sup>loxp/+</sup>, HE) and knockout (Lck-cre<sup>+</sup>Tsc1<sup>loxp/loxp</sup>, KO) mice with tail genomic DNA. (<b>E</b>) Detection of Tsc1 deletion in purified CD8<sup>+</sup>T cells, CD4<sup>+</sup>T cells and non-T cells from spleen of Tsc1 KO mice. Tsc1 was specifically deleted in T cells. (<b>F</b>) Western blot analysis showed efficient deletion of TSC1 and TSC2 protein expression in thymocytes. Phosphorylated S6 level was significantly increased in Tsc1 KO mice.</p
TSC1 is required for naive CD8<sup>+</sup> T cell homeostasis in adoptive transfer mouse models.
<p>Sorted naïve CD8<sup>+</sup>T cells were adoptively transferred into syngeneic Rag1<sup>−/−</sup> or irradiated recipients. The levels of transferred naïve CD8<sup>+</sup>T cells in spleen and pLNs of recipients were determined 7 days after transfer. (<b>A</b>) Schematic representation of adoptive transfer of either WT or Tsc1 KO CD45.2<sup>+</sup> naïve CD8<sup>+</sup> T cells into Rag<sup>−/−</sup> host. (<b>B</b>) One representative staining of CD8<sup>+</sup>T cells in pLN of Rag1<sup>−/−</sup> mice after transfer of sorted naïve CD8<sup>+</sup>T cells. (<b>C</b>) Tsc1 KO CD45.2<sup>+</sup> naïve CD8<sup>+</sup> T cells showed homeostatic defect in spleen and pLNs. Statistic analysis of percentage and numbers of transferred CD8<sup>+</sup>T cells in spleen as well as pLN. (<b>D</b>) Schematic representation of adoptive transfer of mixed(1∶1) population of WT CD45.1<sup>+</sup> and Tsc1 KO CD45.2<sup>+</sup> naïve CD8<sup>+</sup>T cells into Rag<sup>−/−</sup> host. (<b>E</b>) One representative staining of CD45.1 and CD45.2 on gated CD8<sup>+</sup>T cells in pLNs of Rag1<sup>−/−</sup> mice 7 days after transfer of sorted naïve CD8<sup>+</sup>T cells. (<b>F</b>) The ratio between WT CD45.1<sup>+</sup>CD8<sup>+</sup> and Tsc1 KO CD45.2<sup>+</sup>CD8<sup>+</sup> T cells as well as the frequency of CD45.1<sup>+</sup> or CD45.2<sup>+</sup>CD8<sup>+</sup> T cells in spleen and pLNs were summarized. (<b>G</b>) Schematic representation of adoptive transfer of either WT or Tsc1 KO CD45.2<sup>+</sup> naïve CD8<sup>+</sup> T cells into sublethally irradiated(4 Gy) CD45.1<sup>+</sup> host. (<b>H</b>) One representative staining of CD45.1 and CD8 for pLN cells of recipients 7 days after transfer of sorted naïve CD8<sup>+</sup>T cells. (<b>I</b>) Lower percentage and cell number of Tsc1 KO CD45.2<sup>+</sup> naïve CD8<sup>+</sup> T cells in spleen and pLNs. *p<0.05, **p<0.01, and ***p<0.001 compared with WT group. Data were shown as Mean±SD (3–5 mice each group). One representative of two or three independent experiments with identical results was shown.</p