Immunohistochemical staining (dark brown coloring) for Bcl-2 protein in lung specimens from chickens incubated under normal and hypoxic conditions.

<p>Bcl-2 protein expression in lung samples from nW, hT, and hW gradually increased moderately from E16 to E18 before the onset of lung functioning (E–H), and disappeared at E19. By E18 in nW lungs, weaker Bcl-2 expression was observed relative to hT (G) and hW (H) chicken groups. In nT chicken sections, Bcl-2 protein staining was nearly unchanged throughout the developmental stages examined (A, E, K). To see details of the E18 lung structure, higher-magnification photomicrographs were taken. Bcl-2 staining was detected in the mesenchyme (arrows) around the ACs and not in the infundibula or atrias of chicken lung (F’, G’, H’)”. In the hW section at E18, Bcl-2 staining was strong (arrow in H’), but weaker in hT and nW at this stage (arrows in G’, F’). Scale bar  =  200 µm.</p

<i>bcl-2</i> is a target gene of miR-15a, but not miR-16 in chicken lung.

<p>(A) miR-15a regulates the translation of <i>bcl-2</i> mRNA through a sequence that is not conserved with human sequence. miR-15a and the binding site in the gga-<i>bcl-2</i> 3′-UTR are shown, but miR-16 shows no target site in this part of the sequence. (B) miR-15a/16 have a consistent target site in human <i>bcl-2</i> 3′-UTR. The miR-15a binding site in the <i>bcl-2</i> 3′-UTR sequence mediates translation repression by miR-15a. (C) The luciferase reporter vector contains two luciferase cDNAs, Renilla luciferase (hRluc) and firefly luciferase (hluc). The <i>bcl-2</i> 3′-UTR was fused to the hRluc cDNA downstream sequence. In co-transfected cells, the miR-15a mimic decreased the expression of hRluc and miR-15a mimic inhibitor rescued hRluc activity; no differences were seen for miR-16. Data are expressed as the mean ± SEM. * <i>P</i><0.05, ** <i>P</i><0.01.</p

Expression of miRNAs in chicken lung tissues.

<p>(A) Total RNA from tissues from E19 chicken embryos was blotted with probes for miR-15a, miR-144 and U6 (loading control). miR-15a and miR-144 were identified in late-stage embryonic lung tissue. In each sample, the total RNA was mixed with samples from 9 chickens. (B) Quantitative expression analysis of miR-15a showed hypoxia-related expression that was affected by both the species and environmental conditions. The hW chicken group was most sensitive relative to other three groups. In the nW chicken group, there was a response at E19. At E19 of the nW chicken group, the expression of miR-15a remained relatively high in the hT chicken group compared with the nT, nW chicken groups and was largely unchanged in the nT chicken group through the whole embryo stages. Data are expressed as the mean ± SEM for each group. (C) Quantitative expression analysis of miR-15a and miR-16 in the embryonic lung, heart, brain and liver at E16 and E19 tissues and were expressed as the mean ± SEM for each group. Under hypoxia stress, miR-15a was more highly expressed at E19 than at E16 in the brain, heart and lung for the hW group and in the lung and brain for the hT group. miR-16 showed a weak response to stress in the embryonic lung (hW). Result statistically different are indicated with an asterisk/s (* <i>P</i><0.05; ** P<0.01; ns : not significant). E16-20  =  embryonic d13-20, respectively. d1, d2, d3  =  the 1^st day after hatching, the 2^nd day after hatching, the 3^rd day after hatching.</p

Analysis of apoptosis in lung specimens from chickens incubated under normal and hypoxic conditions.

<p>From E16 to E18, no TUNEL staining was identified (A–H). At E19, apoptotic cells were localized in the mesenchyme surrounding the atrias and infundibula of the chicken lungs (L, M, N). There was no obvious staining in nT chicken at E19 (K). At higher magnification, staining was clearly seen in the regions between ACs and not in the parabronchi, atrias, or infundibula (arrows in L’, M’, N’). hW staining at E19 (arrow in N’) was clearly darker than that observed in hT or nW lung sections at this stage (arrows in M’, N’). The tube density in hW at E19 was also higher than that in sections from those other two groups. Scale bar  =  200 µm.</p

HIF-1α and Bcl-2 expression in lung tissue under hypoxia and normoxia conditions.

<p>Data were obtained from hT, hW, nT, and nW chicken tissues at E16, E18, E19, and E20. (A) Quantitative expression analysis of HIF-1α mRNA in embryonic lung at E16, E18, E19, and E20. The stress reaction in hW was robust as compared with the smooth response in hT. Data are expressed as the mean ± SEM for each group. (B) Quantitative expression analysis of <i>bcl-2</i> mRNA in the embryonic lung, heart, brain and liver at E16 and E19. There were no changes in the expression of <i>bcl-2</i> mRNA between the E16 and E19 in each kind of embryonic tissues. Data are expressed as the mean ± SEM for each group. (C) Analysis of HIF-1α and Bcl-2 expression at the protein level in lung tissue (shown in western blot and densitometry value). HIF-1α protein expression increased from E16 to E19, however in different level in hT, hW, nT, and nW group. Bcl-2 protein did show different levels of expression across different time points and different groups. (* <i>P</i><0.05; ** P<0.01; ns  =  not significant).</p

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

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

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

Number of SNPs by region.

<p>Number of SNPs by region.</p