Defective patterning and ossification in <i>Hand1</i> mutant long bones.

<p>(A) Wild-type (a) and <i>Hand1</i>^<i>Tg/+</i><i>; Twist2-Cre</i> mutants (b,c) at P1. (B) <i>Hand1</i>^<i>Tg/+</i><i>; Twist2-Cre</i> mutant (Tg) and wild-type (Wt) littermate at P21. (C) Bone staining of P1 Wt and <i>Hand1</i> mutant forelimbs (top panels) and hindlimbs (bottom panels), as indicated. Scale Bars: 1mm. (D) Bone staining of Wt and <i>Hand1</i> mutants at E16.5, P1, and P10, as indicated. Scale Bars: 1 mm (a,b,d,e), 2 mm (c,f). fl, forelimb; hl, hindlimb; r, radius; u, ulna; hu, humerus; s, scapula; fe, femur; t, tibia; fi, fibula.</p

Hand1 inhibits the <i>Ihh</i> promoter through Runx2 transactivation.

<p>(A) qPCR analysis of <i>Hand1</i> (a), <i>Ihh</i> (b), and <i>Runx2</i> (c) transcripts in ATDC5 cells stably transfected with an empty vector (Control) or <i>Hand1</i> expression vector. (B) Luciferase assays. COS1 cells were transiently cotransfected with pIhh-luc reporter and the indicated expression vectors. Luciferase data (b) is shown as a percentage of Runx2 activation (normalized to 1.0). The data represent the mean ± SD. (C) Model for transcriptional regulation of endochondral ossification by Hand1. Hand1 inhibits Runx2-dependent <i>Ihh</i> expression, which normally promotes Runx2 expression in the perichondrium and the periosteum, which, in turn, is required for osteoblast differentiation.</p

Participation of RelA in Tax1-mediated growth inhibition and apoptosis.

<p>(A) Growing Kit 225 cells were transfected with RelA-specific siRNA and cultured for 72 h. RelA expression was examined by RT-PCR. GAPDH was used as an internal control. (B) Growing Kit 225 cells were transfected with RelA-specific siRNA 24 h before adenovirus infection (Ad-Tax1 or Ad-Con), and harvested 48 h and 72 h post infection for western blotting. RelA, p100, p52 and Tax1 expression was monitored by immunoblotting with anti-RelA, anti-p52 and anti-Tax1 antibodies. β-Tubulin was used as an internal control. (C) siRNA-treated growing or resting Kit 225 cells were infected with Ad-Tax1 or Ad-Con, and cultured for indicated times. Mitochondrial activity was measured by MTT assay. Relative percentages of Ad-Tax1 to Ad-Con are shown. *, <i>p</i><0.05. (D) siRNA-treated growing Kit 225 cells were infected with Ad-Tax1 or Ad-Con, and cultured for 72 h. Tax1 expression and DNA fragmentation were measured by flow cytometry. (E) Percentage average number of cells undergoing apoptosis was calculated from three independent experiments. Values are shown as the means ± SE. *, <i>p</i> < 0.05.</p

Induction of apoptosis by Tax1 in growing cells.

<p>(A) Growing or resting Kit 225 cells were infected with Ad-Tax1 or Ad-Tax2B, and cultured for 72 h. DNA fragmentation was detected by the TUNEL assay using a flow cytometer. The thick line and gray area indicate Ad-Tax1- or Ad-Tax2B-treated cells and Ad-Con-treated cells, respectively. (B) Percentage averages number of the cells undergoing apoptosis was calculated from three independent experiments. Values are shown as the means ± SE. *, <i>p</i> < 0.05. (C) DNA fragmentation and Tax1 expression were measured using a flow cytometer. Tax1 was detected with biotin-labeled anti-Tax1 antibody (Lt-4) and phycoerythrin-conjugated streptavidin, and DNA fragmentation was measured by TUNEL assay.</p

Induction of chemokines and cytokines by Tax1-mediated RelA activation.

<p>(A) Growing or resting Kit 225 cells were infected with Ad-Tax1 or Ad-Tax2B, and cultured for 72 h. The cytoplasmic and nuclear extracts were subjected to western blotting with antibodies for RelA, Tax1, Tax2, β-Tubulin and nucleolin. (B) The reporter plasmid carrying the NF-κB-binding sites was transfected into Kit 225 cells along with the Tax1 and Tax2B expression plasmids (pMT-2Tax and pHβAP-r-1-neoTax2B, respectively). After 48 h of culture with or without IL-2, the cells were harvested for luciferase assay, which was further normalized against protein content. Values are shown as the means ± SE. (C and D) Growing or resting Kit 225 cells were infected with Ad-Tax1 or Ad-Tax2B, and cultured for 72 h (C). siRNA-treated growing and resting Kit 225 cells were infected with Ad-Con or Ad-Tax1, and cultured for 48 h (D). Gene expression was monitored by RT-PCR. 18 S rRNA was used as an internal control.</p

Differential effects of Tax1 on cell proliferation.

<p>Growing (A) or resting (B) Kit 225 cells, PBLs (C) and Jurkat cells (D) were infected with Ad-Tax1, Ad-Tax2B or Ad-Con, and cultured for the indicated times. Mitochondrial activity was measured by the MTT assay and the cells were enumerated. Relative percentages of Ad-Tax1 or Ad-Tax2B samples to Ad-Con samples are shown. *, <i>p</i> < 0.05. Tax expression was monitored by qPCR. Values are shown as the means of the copy numbers ± SE after normalization against 18 S rRNA content.</p

Effects of Tax1 mutants on growth inhibition and apoptosis.

<p>(A and B) Growing or resting Kit 225 cells were infected with Ad-Tax1, Ad-TaxM22, Ad-Tax703, Ad-Taxd17/5 or Ad-Con. After culture for the indicated time period, the mitochondrial activity, cell number and LDH activity of these cells were determined. Relative percentages to Ad-Con are shown. *, <i>p</i> < 0.05. Tax1 mutants expression was monitored by qPCR. Values are shown as the means of the copy numbers ± SE after normalization against 18 S rRNA content. (C) Tax1 expression and DNA fragmentation were measured by flow cytometry by using adenovirus-infected growing Kit 225 cells. (D) Percentage average number of the cells undergoing apoptosis in adenovirus-infected cells was calculated from three independent experiments. Values are shown as the means ± SE. *, <i>p</i> < 0.05.</p

A link between apoptosis and the non-canonical NF-κB pathway.

<p>(A) Kit 225 cells were transfected with p100-specific siRNA and cultured for 48 h. p100 expression was detected by western blotting. β-Tubulin was used as an internal control. (B) Growing Kit 225 cells were transfected with p100-specific siRNA and cultured for 24 h, and infected with Ad-Con or Ad-Tax1, followed by harvest at indicated times. Mitochondrial activity was measured by MTT assay. Relative values of Ad-Tax1 to Ad-Con are shown. *, <i>p</i> < 0.05. (C) Expression of adenovirus-derived Tax1 and its mutant Tax225–232 proteins was measured by immunoblotting with anti-Tax1 antibody (Taxy-7). p100 and p52 levels were detected by anti-p52 antibody, which recognizes both p100 and p52. β-Tubulin was used as an internal control. (D) Growing Kit 225 cells were infected with Ad-Tax1, its mutant Ad-Tax225–232 or Ad-Con, and cultured for indicated times. Mitochondrial activity was measured by MTT assay. Relative percentages of Ad-Tax1 or Ad-Tax225–232 samples to Ad-Con samples are shown. *, <i>p</i> < 0.05. (E) Growing Kit 225 cells were infected with Ad-Tax1 or Ad-Tax225–232, and cultured for 72 h. DNA fragmentation was measured by flow cytometry. The thick line and gray area indicate Ad-Tax1- or Ad-Tax225–232-treated cells and Ad-Con-treated cells, respectively.</p

Cell cycle profiles of growing and resting cells with Tax1.

<p>(A) Growing or resting Kit 225 cells were infected with recombinant adenoviruses expressing Tax1 (Ad-Tax1), Tax2B (Ad-Tax2B) or control virus (Ad-Con), and cultured for 72 h. DNA content was determined using a flow cytometer after PI staining. (B) Percentages of cell cycle phases were calculated from Fig 1A. M1, M2, M3, M4 and M5 indicate the sub-G0, G0/G1, S, G2/M and multinuclear cells, respectively.</p

Induction of cell cycle regulatory genes by Tax1 in resting cells.

<p>(A) Growing or resting Kit 225 cells were infected with Ad-Tax1 or Ad-Tax2B. Cells were cultured for 72 h and harvested for RNA isolation. The levels of CDK4, cyclin D2, CDK2 and cyclin E mRNA were measured by qPCR. Values are shown as the means ± SE after normalization against 18 S rRNA content. *, <i>p</i> < 0.05. (B) Reporter plasmids carrying binding sites for the wild-type and point-mutated E2F were transfected into Kit 225 cells along with Tax1 and Tax2B expression plasmids (pMT-2Tax and pHβAP-r-1-neoTax2B, respectively). After 48 h of culture with or without IL-2, the cells were harvested for luciferase assay, which was subsequently normalized against protein content. Values are shown as fold activation relative to luciferase activity of cells with pHβAP-r-1-neo (means ± SE).</p