Knockdown of IAN5 in 23–1–8 T Lymphocytes

<div><p>(A) 23–1–8 T lymphocyte clones expressing shRNAs were analyzed for <i>IAN5</i> mRNA expression and cultured in the presence or absence of IL-2. Cell viability was quantified by PI staining and flow cytometry analysis. </p> <p>(B and C) Cells in 48-h culture were analyzed for apoptosis induction. Frequencies of Annexin-V-positive cells (B) or mitochondrial membrane potential (Δψm)-negative cells (C) are shown.</p> <p>(D) 23–1–8 T cells expressing shRNAs with or without human <i>Bcl-xL</i> were analyzed for <i>IAN5</i> expression by quantitative RT-PCR and for human <i>Bcl-xL</i> expression by conventional RT-PCR (left panel). Cells were cultured in the presence or absence of IL-2 for 72 h or in the presence of IL-2 and 5 μM helenalin for 48 h, and cell viability was quantified by PI staining (right panel). </p> <p>Graphs show means ± standard errors.</p> <p>NS, not significant ( <i>p</i> ≥ 0.05);^**<i>p</i> < 0.01. </p></div

Knockdown of IAN1, IAN4, and IAN5 in Thymocyte Development

<div><p>(A) Diagram of retroviral shRNA constructs.</p> <p>Puro <i>^r, </i> puromycin resistance gene. </p> <p>SIN-LTR, self-inactivating long terminal repeat.</p> <p>(B) BW5147 cells expressing IAN1-HA, IAN4-HA, or IAN5-HA were infected with shRNA retroviruses, and the infected cells were enriched by puromycin selection. Protein expression levels were analyzed by anti-HA IB. <i>Luciferase</i> (Luc) shRNA was used as control. </p> <p>(C) Day 14.5 fetal thymocytes infected with shRNA retroviruses were reconstituted in FTOC. EGFP⁺ cells purified on day 6 were analyzed for mRNA expression. </p> <p>(D) Viable cell numbers of total cells (striped bars) and EGFP⁺ cells (open bars) in FTOC on day 6. </p> <p>(E) EGFP histograms of total cells and CD4/CD8 profiles of EGFP⁺ cells in FTOC on day 6. The frequency of EGFP⁺ cells and the mean fluorescence intensity (MFI) in the indicated area are shown in the histograms. Numbers in dot plots show the frequency of EGFP⁺ cells within boxes. </p> <p>(F) Frequencies of indicated cell populations on day 6.</p> <p>Filled bars indicate significant difference from the values in the control group ( <i>Luc</i> shRNA) ( <i>p</i> < 0.05). </p> <p>Bar graphs show means ± standard errors.</p> <p>NS, not significant ( <i>p</i> ≥ 0.05);^*<i>p</i> < 0.05;^**<i>p</i> < 0.01. In (D) and (F), <i>Luc</i> shRNA, <i>n</i> = 11; <i>IAN1</i> shRNA, <i>n</i> = 5; <i>IAN4</i> shRNA, <i>n</i> = 11; <i>IAN5</i> shRNA, <i>n</i> = 8. No significant difference in the CD4/CD8 developmental profiles was observed in EGFP⁻ cell populations. </p></div

Interaction of IAN Family Proteins with Bcl-2 Family Proteins

<div><p>(A) 293T cells were co-transfected with FLAG-tagged IAN molecules together with Bcl-2, Bcl-xL, HA-tagged Bax, HA-tagged Bak, HA-tagged Bad, BimEL, HA-tagged IκBα, or EGFP. Cell lysates were IP with anti-FLAG M2 antibody and IB with indicated antibodies.</p> <p>(B) 23–1–8 T cells expressing EGFP alone (Vector), FLAG-tagged IAN4, or FLAG-tagged IAN5 were IP with anti-FLAG M2 antibody and IB with anti-Bcl-2 or anti-Bcl-xL antibody.</p> <p>(C) 23–1–8 T cells expressing FLAG-tagged IAN4 or FLAG-tagged IAN5 were IP with normal IgG or anti-Bcl-2 or anti-Bcl-xL antibody and IB with anti-FLAG M2 antibody. Arrows indicate FLAG-tagged IAN4 or FLAG-tagged IAN5.</p> <p>(D) 23–1–8 T cells expressing EGFP alone (Vector), FLAG-tagged IAN4, or FLAG-tagged IAN5 were cultured in the presence or absence of IL-2 for 36 h. Cell lysates were IP with anti-FLAG M2 antibody and IB with anti-Bax antibody.</p> <p>(E) Nuclear and heavy membrane fractions prepared from 23–1–8 T cells were lysed in buffer containing 1% CHAPS. The lysates were IP with normal rabbit IgG or anti-IAN4 antibody and IB with anti-Bcl-2 antibody. Means and standard errors ( <i>n</i> = 4) of relative intensities of the bands were analyzed by using NIH Image software. </p> <p>^*<i>p</i> < 0.05;^**<i>p</i> < 0.01. </p></div

Expression of Mouse IAN Family Genes

<div><p>(A) Quantitative RT-PCR analysis of total RNA from C57BL/6 mouse tissues, purified splenocyte subsets, and purified thymocyte subsets. The mRNA levels of IAN family genes were initially normalized to <i>GAPDH</i> levels, and were further normalized to the levels expressed in the thymus. Relative expression of all IAN family genes in the thymus tissue is indicated as 1. </p> <p>(B) Relative mRNA levels of <i>IAN1, IAN4,</i> and <i>IAN5</i> in CD4⁺CD8⁺, CD4⁺CD8⁺CD5^low, and CD4⁺CD8⁺CD5^high thymocytes from C57BL/6 (wild-type) mice and CD4⁺CD8⁺ thymocytes from TCRα-deficient mice [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040103#pbio-0040103-b042" target="_blank">42</a>]. </p> <p>(C) <i>IAN1, IAN4,</i> and <i>IAN5</i> mRNA levels in CD4⁺CD8⁺ thymocytes from positive selector (AND-TCR Aβ^+/+ and 2C-TCR^k/b [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040103#pbio-0040103-b043" target="_blank">43</a>]) TCR-transgenic mice and null selector (AND-TCR Aβ^−/− [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040103#pbio-0040103-b044" target="_blank">44</a>] and 2C-TCR^k/k) TCR-transgenic mice. </p> <p>(D) Relative mRNA levels of <i>IAN1, IAN4,</i> and <i>IAN5</i> in total, CD4⁺CD8⁺CD69^low, CD4⁺CD8⁺CD69^high, CD4⁺CD8⁻CD69^high and CD4⁺CD8⁻CD69^low thymocytes from C57BL/6 mice. </p> <p>(E) Thymocytes from TCRα-deficient mice were cultured with or without phorbol 12-myristate 13-acetate (0.2 ng/ml) and ionomycin (0.2 μg/ml) for the indicated periods.</p> <p>(F) Thymocytes from Aβ^−/− β2m^−/− mice [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040103#pbio-0040103-b045" target="_blank">45</a>] were cultured with or without plate-bound anti-CD3 (clone 2C11) and anti-CD28 (clone 37.51) antibodies for 24 h. </p> <p>Bar graphs show means ± standard errors.</p></div

IAN Family Genes

<div><p>(A) The cluster of IAN family genes in the genome of indicated species. Mouse IAN genes and their orthologs in human and rat are indicated. For chicken, zebrafish, and thale cress, arrows indicate genes that putatively encode AIG1-domain-containing proteins. In chicken, 19 genes are predicted to encode AIG1 domain–containing proteins, and arrows indicate 15 genes clustered on Chromosome 2. In zebrafish, a cluster of 23 genes was found on Chromosome 16. In thale cress, ten out of 14 predicted genes are clustered on Chromosome 1.</p> <p>(B) Predicted structures of mouse IAN family proteins. Numbers refer to amino acid residues of full-length proteins.</p> <p>(C) A neighbor-joining tree of the AIG1 domain of IAN proteins. A. thaliana AIG1, residues 44–243; N. tabacum NTGP4 (AAD09518), residues 23–222; G. max NTGP4 (BI316235), residues 1–118; O. sativa AIG1 (CAE04223), residues 31–230; <i>Z. mays</i> AIG1 (AW120061), residues 1–200; D. rerio IAN (BC053197), residues 1–200; G. gallus IAN (XP_427942), residues 3–202; M. musculus IAN1, residues 31–230; R. norvegicus IAN1, residues 31–230; H. sapiens IAN1, residues 45–244. No IAN genes were found in the genomes of Drosophila melanogaster (fly), Anopheles gambiae (mosquito), Ciona intestinalis (sea squirt), Caenorhabditis elegans (nematode), Saccharomyces cerevisiae (yeast), and all bacteria and archea. </p></div