Lats2-mediated repressed proliferation of 3T3L1 preadipocytes.

<p>(A) TEAD3 is the main TEAD expressed in 3T3L1 cells, fat and liver tissue. RT-PCR assay was performed using TEAD1–4 specific primers. (B) TEAD3 localizes to the nucleus, but YAP and TAZ remain in the cytoplasm due to phosphorylation by Lats2. Micrographs depict TEAD3 in 3T3L1 cells as detected by anti-TEAD3 antibody (green). Anti-YAP and anti-TAZ antibodies appear red. The nucleus was stained by DAPI (blue). The scale bar represents 20 µm. (C) Lats2-mediated decrease of Hippo target gene expression at the mRNA level. Target gene transcript levels were measured by quantitative RT-PCR. The data shown are the means+S.D. of three independent experiments. (D) Lats2-mediated decrease of Hippo target gene expression at the protein level. (E) Preadipocytes growth is inhibited by Lats2. Cells were cultured in 96-well culture plates and treated with MTS at the designated times (every 24 h). After incubation, the absorbance was recorded at 490 nm. (F) Preadipocytes proliferation is delayed by Lats2. Cells were cultured in 96-well culture plates and treated with BrdU at the designated times (every 24 h). After incubation with BrdU antibody and substrate, the absorbance was read at 450 nm. (G) Lats2-mediated less DNA synthesis of preadipocytes. Micrographs show the BrdU incorporated in 3T3L1 cells as detected by anti-BrdU antibody (green). Cell nuclei were stained by DAPI (blue). The scale bar represents 20 µm. (H) Cell cycle progression of preadipocyte is delayed by Lats2. Cells were cultured in 10-cm dishes for 48 h and then stained by PI for flow cytometry. Statistics from three separate experiments showing the percentages of cells in G₁, G₂ and S phase, respectively. In (C), (E), (F) and (H), <i>P</i>-values were calculated using the Student’s t-test (*, <i>P</i><0.05; **, <i>P</i><0.01).</p

YAP and TAZ are phosphorylated by Lats2 and accumulate in the cytoplasm.

<p>(A) Lats2-mediated enhanced phosphorylation of YAP and TAZ. Whole-cell lysates were prepared from Lats2-transfected cells, immunoblotted with Lats2, p-YAP, YAP, p-TAZ, TAZ and Tubulin antibodies. (B) Lats2-mediated enhanced cytoplasmic accumulation of YAP and TAZ. Micrographs depict YAP and TAZ in 3T3L1 cells, as detected by anti-YAP and anti-TAZ antibodies (green). The nucleus was stained by DAPI (blue). The scale bar represents 20 µm.</p

Adipocyte differentiation is promoted by Lats2.

<p>(A) Western blot analyses. Whole-cell lysates were prepared from differentiating 3T3L1 cells (day 0-day 8). (B) Lats2 enhances the transcriptional activity of PPARγ. Cells were co-transfected with pGL3-Basic-aP2-Promoter plasmids, pcDNA3.1-PPARγ plasmids or pcDNA3.1 empty vectors and pRL-TK vectors (<i>pRenilla</i> as internal control). After 24 h, cells were treated with or without Rosiglitazone (10 µM). pcDNA3.1 denotes pcDNA3.1 empty vector transfection, and PPARγ denotes pcDNA3.1-PPARγ transfection. (C) Lats2-mediated enhanced mRNA levels of SREBP1, PPARγ and its target genes. The data shown are the means+S.D. of three independent experiments. (D) Lats2-mediated enhanced protein levels of SREBP1, PPARγ and its target genes. (E) The differentiation of 3T3L1 cells is accelerated by Lats2. At day 4 and day 8 of adipocyte differentiation, 3T3L1 cells were observed under a microscope. At day 8, cells were stained with Oil Red O and photographed. The scale bar represents 20 µm. (F) and (G) Lats2-mediated enhanced expression of adipocyte marker genes in differentiating 3T3L1 cells. Total RNA and protein were isolated from the cells shown in (E) at day 4 for quantitative RT-PCR and Western blotting, respectively. In (B), (C), (E) and (F), <i>P</i>-values were calculated using the Student’s t-test (*, <i>P</i><0.05; **, <i>P</i><0.01).</p

Lats2 inhibits Wnt signaling.

<p>(A) The protein levels of p-DVL2, β-catenin and Wnt signaling targets decrease during adipocyte differentiation. Total cell lysates were prepared from differentiating 3T3L1 cells. (B) Lats2-mediated decrease in β-catenin level. Western blot shows that the levels of DVL2 phosphorylation and β-catenin protein are both reduced by Lats2. (C) Lats2 suppresses the Wnt3a-induced activity of the TOPflash reporter. TOPflash and pRL-TK plasmids were co-transfected into Lats2-transfected cells and the two control (Vector and Control) cells, which were then treated with or without Wnt3a (50 ng/ml). The pRL-TK plasmid (<i>pRenilla</i>) was used as an internal control. The FOPflash assay was used as a negative control. NM, normal medium. (D) and (E) Lats2 inhibits Wnt target gene expression. Quantitative RT-PCR results from Lats2-transfected cells indicate that Pref-1, LEF1, cyclin D1, and c-Myc mRNA levels are reduced by Lats2. The data shown are the means+S.D. of three independent experiments. Western blot shows that the protein levels of cyclin D1, c-Myc, LEF1 and Axin are also reduced by Lats2. In (C) and (D), <i>P</i>-values were calculated using the Student’s t-test (*, <i>P</i><0.05; **, <i>P</i><0.01).</p

Three IgH isotypes, IgM, IgA and IgY are expressed in Gentoo penguin and zebra finch

<div><p>Previous studies on a limited number of birds suggested that the IgD-encoding gene was absent in birds. However, one of our recent studies showed that the gene was definitely expressed in the ostrich and emu. Interestingly, we also identified subclass diversification of IgM and IgY in these two birds. To better understand immunoglobulin genes in birds, in this study, we analyzed the immunoglobulin heavy chain genes in the zebra finch (<i>Taeniopygia guttata</i>) and Gentoo penguin (<i>Pygoscelis papua</i>), belonging respectively to the order Passeriformes, the most successful bird order in terms of species diversity and numbers, and Sphenisciformes, a relatively primitive avian order. Similar to the results obtained in chickens and ducks, only three genes encoding immunoglobulin heavy chain isotypes, IgM, IgA and IgY, were identified in both species. Besides, we detected a transcript encoding a short membrane-bound IgA lacking the last two CH exons in the Gentoo penguin. We did not find any evidence supporting the presence of IgD gene or subclass diversification of IgM/IgY in penguin or zebra finch. The obtained data in our study provide more insights into the immunoglobulin heavy chain genes in birds and may help to better understand the evolution of immunoglobulin genes in tetrapods.</p></div