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

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

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

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

Cell-penetrating peptide-driven Cre recombination in porcine primary cells and generation of marker-free pigs

<div><p>Cell-penetrating peptides (CPPs) have been increasingly used to deliver various molecules, both <i>in vitro</i> and <i>in vivo</i>. However, there are no reports of CPPs being used in porcine fetal fibroblasts (PFFs). The increased use of transgenic pigs for basic research and biomedical applications depends on the availability of technologies for efficient genetic-modification of PFFs. Here, we report that three CPPs (CPP5, TAT, and R9) can efficiently deliver active Cre recombinase protein into PFFs via an energy-dependent endocytosis pathway. The three CPP<b>–</b>Cre proteins can enter PFFs and subsequently perform recombination with different efficiencies. The recombination efficacy of CPP5<b>–</b>Cre was found to be nearly 90%. The rate-limiting step for CPP<b>–</b>Cre-mediated recombination was the step of endosome escape. HA2 and chloroquine were found to improve the recombination efficiency of TAT<b>–</b>Cre. Furthermore, we successfully obtained marker-free transgenic pigs using TAT<b>–</b>Cre and CPP5<b>–</b>Cre. We provide a framework for the development of CPP-based farm animal transgenic technologies that would be beneficial to agriculture and biomedicine.</p></div

Generation of marker-free Monoallelic <i>MSTN</i> gene knockout pigs using TAT–Cre.

<p>(A) PCR-based genotyping scheme to differentiate the three <i>MSTN</i> alleles: WT, <i>MSTN</i>^+/−with the <i>Neo</i>, and marker-free <i>MSTN</i>^+/−. P3 and P4 primers designed to amplify 788-bp and 370-bp fragments from marker-free pigs. The 788-bp fragment could only be amplified from WT and <i>MSTN</i>^+/- pigs with a 30-s extension time. (B) Identification of marker-free <i>MSTN</i>^+/−piglets using genomic PCR method. WT, wild-type; H₂O, negative control; M^+/-, <i>MSTN</i>^+/−piglets containing <i>Neo</i>; 1<b>–</b>12 are marker-free <i>MSTN</i>^+/−piglets. (C) PCR sequencing analysis of the marker free <i>MSTN</i>^+/−piglets. The 370-bp product is a specific fragment for the marker-free pig genome including one loxP site between P3 and P4. (D) A live marker-free <i>MSTN</i>^+/−pig.</p

Effects of Lysosomotropic agents on TAT–Cre recombination efficiency and toxicity.

<p>Effects of different concentrations of HA2 (A) and chloroquine (B) on TAT<b>–</b>Cre-mediated recombination of a loxP-<i>Neo</i>-loxP target, using the standard curve method (39). Percentage of living cells (as an indication of potential toxicity) in porcine fetal fibroblasts treated with different concentrations of HA2 (C) and chloroquine (D). Error bars, s.d. (n = 3).</p

CPP-mediated Cre recombinase transduction efficiency at 4°C and 37°C, and localization of the CPP–Cre proteins.

<p>(A-C) Transduction efficiencies of CPP<b>–</b>Cre proteins at 4°C and 37°C. Flow cytometry analysis of cells treated for 3 h at 37°C and 4°C at the indicated concentrations of CPP<b>–</b>Cre labeled with Alexa Fluor 488. The cells were washed thoroughly with PBS and then trypsinized to remove the extracellular CPP fusion proteins. (D) Confocal images showing endosomal colocalization of CPP<b>–</b>Cre-488 proteins and FM4-64 (fluorescent, general endosomal marker) in live cells. Scale bar, 10 μm.</p

Design of expression cassettes, purification of the three CPP–Cre proteins, and assessment of their activities in an <i>in vitro</i> assay.

<p>(A) Schematic description of the three CPPs<b>–</b>Cre expression constructs. All the constructs encode Cre recombinase with a His-tag (represented by blue and green boxes, respectively). Red boxes represent CPPs (RQRRKKRG): R9 (RRRRRRRRR), TAT (YGRKKRRQRRR), and CPP5 (KLVPM). (B-D) SDS-PAGE analysis of the purification of CPP<b>–</b>Cre proteins. M, marker; SF, supernatant fraction; PR, precipitation; Ni, Nickel column; G25, G25 column. (E) Schematic of recombination <i>in vitro</i>. The assay substrate, pDFR, was linearized by digestion with <i>Bsa</i>I and was used to assess the recombinase activity of the purified Cre proteins. (F-H) Activity of CPP<b>–</b>Cre proteins <i>in vitro</i>. Reactions were carried out in a 50-μL volume with 300 ng of pDFR and New England Biolabs Cre buffer. Different amounts of purified Cre were added, and the mixtures were incubated at 37°C for 30 min. The reactions were then split in half and resolved on a 1% agarose gel. Lane 1 kb ladder; lane 2, NEB Cre; lane 3, 50 ng Cre; lane 4, 100 ng Cre; lane 5, 150 ng Cre. Note the appearance of four bands including the recombined circular plasmid (migrating at the 2.6 kb pC), the nicked recombined circular plasmid pC-N, the rejoined 5.7 kb “stuffer fragment” pL, and the 8.3kb pDFR-L.</p

CPP–Cre mediated recombination efficiencies in porcine fetal fibroblasts (PFFs).

<p>Structure of the recombination substrate in loxP-Neo-loxP porcine fetal fibroblasts. The loxP-Neo-loxP PFFs, which were generated from the heterozygous <i>MSTN</i>^<i>+/−</i>knockout pig generated previously in our laboratory, contains a single copy of Neo flanked by loxP sites, such that Cre-mediated recombination would remove Neo and the 600 bp fragment cannot be amplified using primers P1 and P2.(B-D) A standard curve for estimating the CPP–Cre recombination frequencies (39).Genomic DNA isolated from cells treated with CPP-Cre were serially diluted in a buffer and subjected to PCR analysis in a reaction volume of 25 μL. A standard curve for the estimation of deletion frequencies was generated. A plasmid containing the sequence of the PCR product corresponding to the genomic recombination induced by Cre was serially diluted in a solution containing genomic DNA isolated from pig fibroblast cells and the diluted samples were subjected to PCR analysis. The intensities of DNA bands corresponding to the recombination event were measured and plotted against the dilution factors. At high values, the band intensities reached a plateau and thus were excluded when the standard curve was plotted. Error bars, s.d. (n = 3).</p