Figure 1. Expression of TRIP6 in embryonic and adult mouse brains.
(A) NIH3T3 cells were transfected with control scrambled shRNA or shTRIP6. Sixteen hours after transfection cells were starved with 0.1%
bovine serum albumin (BSA) containing medium for overnight, and treated with 10% FBS medium for 15 minutes to induce focal adhesion formation. Cells were then fixed and subjected to immunofluorescence of TRIP6 (red) or control mouse-IgG. Nuclei were stained by DAPI (blue).
Arrows indicate TRIP6-positive focal adhesions. The cell surrounded by
60
white dashed lines (C1) is shTRIP6-transfected cell, and the other cell surrounded by blue ones (C2) is an untransfected one. Scale bar=10 mm.
(B) The knockdown efficiency of shTRIP6 was also confirmed by immunoblotting from 3T3 cells. β-actin served as a loading control. (C) Total RNA was extracted from E16.5 and adult mouse forebrains. TRIP6 mRNA was detected by RT-PCR with murine TRIP6-specific primers.
(D) Proteins were extracted from E15.5 and adult mouse forebrains and Western blot analysis of TRIP6 was performed. GAPDH served as a loading control.
61
Figure 2. TRIP6 is expressed by NSCs in the E16.5 mouse forebrain.
Forebrain sections of E16.5 mice were labeled with anti-TRIP6 (green) and anti-SOX2 (red; A-C) or anti-Ki67 (red; D). Nuclei were stained by DAPI (blue). *, staining artifact. (A) Low-magnification images show the
62
staining of TRIP6 and SOX2 in the VZ. (B-C) Images with higher magnification of the dorsal VZ (B) and the ventral VZ (C) from A. (D) Staining of TRIP6 and Ki67 in the dorsal VZ. Arrows: TRIP6 and Ki67 double-positive cells (outlined). (E) Quantification of the percentage of TRIP6-labeled cells that also express SOX2 and Ki67 from B, C and D, respectively. A rectangular area in the VZ (380 x 80 mm2) in each of the 2-mm-thick confocal sections was used for cell counting. About 600 TRIP6-positive cells were counted for each double staining (SOX2 or Ki67) per embryo (n=3). The percentage of double-positive cells over TRIP6-positive cells is shown as mean ± SEM. Scale bar=40 mm for A and 20 mm for B–D.
63
Figure 3. NSCs in the adult SVZ express TRIP6. (A) Adult SVZ
sections were labeled with TRIP6 (green) and SOX2 (red) antibodies. (B) Adult SGZ sections were labeled with TRIP6 antibodies (green). (C–I)
64
Adult SVZ sections were double labeled with TRIP6 (green) and SOX2 (red; C, D), Ki67 (red; E, F), or S100β (red; G, H) antibodies. (D, F, H) Higher magnification images of C, E, and G, respectively. (D’) Another set of higher magnification images of TRIP6 and SOX2 staining in the dorsal VZ. Nuclei were stained by DAPI (blue). Arrows: Double-labeled cells. (I) Quantification of the percentage of TRIP6-labeled cells that also express SOX2, Ki67, or S100β. A rectangular area in the SVZ (380 x 160 mm2) in each of the 2-mm-thick confocal sections were used for cell counting. About 500–800 TRIP6-positive cells were counted for each double staining (SOX2, S100β, or Ki67) per mouse (n=3). The
percentage of double-positive cells is shown as mean ± SEM. LV, lateral ventricle; ST, striatum. Scale bar=50 mm for A and B; 15 mm for C, E, and G; 10 mm for D, D’, F, and H.
65
Figure 4. TRIP6 is not expressed by neurons, astrocytes, and
microglia in adult mouse forebrains. Adult mouse forebrain sections were labeled with TRIP6 (green), and DCX (red; A), MAP2 (red; B), GFAP (red; C), or Iba1 (red; D) antibodies. Nuclei were stained by DAPI (blue). (A) In the SVZ, DCX-positive neuroblast (arrowheads) did not express TRIP6 (arrows). (B) TRIP6 was expressed in the SVZ (arrows), but not by MAP2-positive neurons (arrowhead) in the striatum. (C) In the
66
corpus callosum, GFAP-positive astrocytes (arrowheads) did not express TRIP6 (arrows). (D) In the SVZ, Iba1-positive microglia (arrowheads) did not express TRIP6 (arrows). Scale bar=10 mm for A, C, D and 20 mm for B.
67
Figure 5. TRIP6 is necessary and sufficient for self-renewal of
postnatal NSCs. P7 NSCs were isolated from SVZ and cultured to form 1’ NS. (A–C) 1’ NS were dissociated and electroporated with scrambled shRNA (Ctrl) or shTRIP6 together with GFP plasmids and cultured to form 2’ NS for 5 days. (A) 2’ NS in the control group (arrows). (B) shTRIP6 transfected cells (arrows) did not form 2’NS. C: Quantification of the 2’ NS number. Statistical analysis was performed by t-test, n=3.
(D–F) 1’ NS were dissociated and electroporated with control or TRIP6 together with GFP plasmids and cultured to form 2’ NS for 5 days. (D) 2’
NS in the control group (arrows). (E) TRIP6-positive 2’ NS (arrows) appeared to be larger than those in the control group. (F) Quantification of the 2’ NS diameter. GFP-positive 2’ NS were categorized according to the diameter. Histograms of Ctrl and TRIP6 group were plotted side by side. Statistical analysis was performed by Wilcoxons ranksum test, n=3.
*p<0.05; **p<0.01 compared to the control group. Scale bar=200 mm for A, B, D, and E.
68
Figure 6. TRIP6 is necessary and sufficient for proliferation of
postnatal neural progenitor cells. P7 NSCs were isolated from SVZ and cultured to form 1’ NS. (A–C) 1’ NS were dissociated and transfected with scrambled shRNA (Ctrl) or shTRIP6 together with GFP plasmids and cultured in differentiation condition for one day. BrdU was added 2 hr before fixation. Cells were labeled with GFP (green) and BrdU (red) antibodies. Nuclei were stained by DAPI (blue). Arrows represent GFP and BrdU double-positive cells. (C) Quantification of A and B. n=4. (D–
F) 1’ NS were dissociated and transfected with control or TRIP6 together with GFP plasmids and cultured in differentiation condition for 2 days.
BrdU was added 2 hr before fixation. (F) Quantification of D and E. For normalization, the value of the control group was set as 100 ± 0% and the value from the experimental group was shown as the percentage of the control group. n=3. Statistical analysis was performed by t-test, *p<0.05 compared to the control group. Scale bar=50 mm for A, B, D, and E.
69
Figure 7. TRIP6 inhibits differentiation of postnatal NSCs. P7 NSCs were isolated from SVZ and cultured to form 1’ NS. (A–E) 1’ NS were dissociated and transfected with scrambled shRNA (Ctrl) or shTRIP6 along with GFP constructs, cultured in differentiation condition for 3 days, and immunostained with GFP (green), Tuj1 (red in A, B, F, G), or GFAP (red in C, D, H, I) antibodies. Nuclei were stained by DAPI (blue).
Arrows represent GFP and Tuj1 (or GFAP) double-positive cells. (E) Quantification analysis of A–D. (F–J) 1’ NS were dissociated and transfected with control (Ctrl) or TRIP6 along with GFP expression constructs, cultured in differentiation condition for 3 days, and
immunostained with GFP, Tuj1, or GFAP antibodies. (J) Quantification analysis of F–I. The percentage of double-positive cells in transfected cells is shown as mean ± SEM. For normalization, the value of the
control group was set as 100% and the value from the experimental group
70
was shown as the percentage of the control group. t-test, n=3. *p<0.05;
**p<0.01 compared to the control group. Scale bar=50 mm.
71
Figure 8. TRIP6 activates the Notch pathway. In the Luciferase reporter assay of Notch signaling pathway, P19 cells (A) or SH-SY5Y cells (B) were transfected with control (Ctrl) or TRIP6 along with firefly Luciferase reporter constructs containing either wild-type (WT) or mutant (MT) CBF binding sites. US2-renilla Luciferase constructs were
co-transfected to serve as internal controls. Cells were lysed one day after transfection. Normalized luciferase activities are shown as mean ± SEM.
t-test, n=3. *p<0.05 compared to the control group.
72
Chapter three: YAP Mediates TRIP6-Promoted Neural Stem Cell Maintenance in the Postnatal Mammalian
Subventricular Zone-Olfactory Pathway
Author Contributions
Yun-Ju Laiand Jui-Chen Tsai performed experiments and analyzed data of Figure 9 and Figure 10A.
Ming-Yang Li designed and performed experiments and analyzed data of cell cultures and biochemical experiments in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 10B-C, Figure 11, Figure 12 and wrote the manuscript.
Jenn-Yah Yu designed experiments and wrote the manuscript.
Tsu-Wei Wang designed all experiments and wrote the manuscript.
73
1. Abstract
In the adult mammalian brain, new neurons are continuously generated from neural stem cells (NSCs) in the subventricular zone (SVZ)-olfactory bulb (OB) pathway. YAP, a transcriptional co-activator of the Hippo pathway, promotes cell proliferation and inhibits differentiation in embryonic neural progenitors. However, the role of YAP in postnatal NSCs remains unclear. Here, we showed that YAP was detected in NSCs of the postnatal mouse SVZ. Forced expression of Yap promoted NSC maintenance and inhibited differentiation, whereas depletion of Yap by RNA interference or conditional knockout blocked NSC maintenance and induced neuronal differentiation. Furthermore, thyroid hormone receptor interacting protein 6 (TRIP6) recruited protein phosphatase PP1A to dephosphorylate LATS1/2, therefore inducing YAP nuclear localization and activation. Moreover, TRIP6 promoted NSC maintenance, cell proliferation and inhibited differentiation through YAP. Together, our findings demonstrate a novel interaction between YAP and TRIP6, which is critical for regulating postnatal neurogenesis in the SVZ-OB pathway.
74
2. Introduction
Neurogenesis occurs throughout life in the subventricular zone (SVZ)-olfactory bulb (OB) pathway and the hippocampal dentate gyrus (DG) in the mammalian brain (Ming and Song, 2011). During adult neurogenesis, neural stem cells (NSCs) generate transit-amplifying cells, which give rise to neuroblasts. Neuroblasts born in the SVZ or subgranular zone (SGZ) of the DG then migrate along the rostral migratory stream (RMS) to the OB or radially into the DG, respectively (Ming and Song, 2011).
Upon arrival at their destinations, neuroblasts differentiate into mature neurons and incorporate into neural circuits (Ming and Song, 2011). New neurons in the OB play important roles in odor discrimination and
perceptual learning (Moreno et al., 2009). In the DG, new neurons are required for not only hippocampal-dependent learning and memory, but also the therapeutic effect of anti-depressants (Deng et al., 2010;
Santarelli et al., 2003). Environmental and physiological conditions, as well as intrinsic signaling networks, have been demonstrated to regulate adult neurogenesis (Ming and Song, 2011). However, how various
intricate signalings coordinate to control adult neurogenesis remains to be studied.
The Hippo pathway is important for regulating organ size and cell proliferation (Zhao et al., 2011). Upon activation of the Hippo pathway, MST1/2 phosphorylate and activate LATS1/2. Activated LATS1/2 phosphorylate and inhibit the transcriptional co-activator YAP (Zhao et al., 2011). When the Hippo pathway is not activated, unphosphorylated YAP is transported into the nucleus and interacts with the transcription
75
factor TEAD to induce the expression of target genes involved in cell proliferation (Cao et al., 2008; Zhao et al., 2011). YAP has been shown to promote cell proliferation and inhibits differentiation in mouse embryonic stem cells, the developing chicken spinal cord, rodent retinal progenitor cells, ependymal progenitors and cortical progenitor cells (Cao et al., 2008; Lian et al., 2010; Lin et al., 2012; Park et al., 2016; Zhang et al., 2012). These studies suggest that the Hippo pathway plays a crucial role in regulating cell differentiation. While the role of the Hippo pathway in regulating stem cell properties is emerging, whether the Hippo pathway affects self-renewal or multi-potency of postnatal NSCs remains elusive.
In addition, as a new signaling pathway in growth control, novel regulators of the Hippo pathway remain to be identified.
Thyroid hormone receptor interacting protein-6 (TRIP6) belongs to the Zyxin family, which contains a proline-rich region in its amino-terminus and three LIM domains in the carboxy-terminus (Lin and Lin, 2011;
Rauskolb et al., 2011). Through the three LIM domains, TRIP6 interacts with a number of proteins to regulate cell motility, proliferation, survival and transcription (Lai et al., 2010; Rauskolb et al., 2011). We recently report that TRIP6 promotes NSC maintenance in the postnatal SVZ (Lai et al., 2014). Interestingly, another LIM domain protein AJUBA has been shown to regulate the Hippo pathway by binding to LATS1 and activating YAP (Sun and Irvine, 2013). Whether TRIP6 interacts with the Hippo pathway in regulating NSC maintenance is still unknown.
In this study, we report that YAP is both necessary and sufficient for maintaining NSCs in the postnatal SVZ. Furthermore, TRIP6 regulates
76
postnatal NSC properties through disinhibiting YAP. These findings provide a novel mechanism of the Hippo pathway in regulating postnatal neurogenesis.
77
3. Materials and methods
3.1 AnimalsMice procedures were performed in accordance to guidelines and
protocols approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan Normal University (Approval Number 101026). Transgenic mice were maintained in C57BL/6J background and were exposed to photoperiod (12L: 12D) in an IVC system with
unlimited food and water. Cre recombinase-mutant estrogen receptor fusion protein (CreER) was under the control of SVZ NSC-specific Nestin enhancer (Nestin-CreER mice; (Burns et al., 2007)). LoxP-floxed Yap (Yapf/f) mice (Zhang et al., 2010b) were crossed with Nestin-CreER mice. Conditional knockout of Yap in SVZ NSCs was induced by
tamoxifen treatments. 6~8-week-old mice were injected with tamoxifen (133 mg/kg body weight; Sigma-Aldrich) dissolved in corn oil
intraperitonealiy (i.p.) daily for five days. Pulse BrdU (100 mg/kg body weight; Sigma-Aldrich; dissolved in phosphate buffered saline (PBS)) labeling was administered twice and six hours apart to label mitotically active cells at week one, five or nine after the last tamoxifen injection. 14 days after BrdU injections, animals were sacrificed and processed for immunofluorescence.
3.2 Fixation and sectioning
For perfusion, animals were deeply anesthetized by an i.p. injection of Avertin solution (0.025 g of 2, 2, 2-tribromomethylalcohol and 0.025 ml of 2-methyl-2-butanol in 0.975 ml water; 17 ml/kg body weight) and
78
perfused with 0.9% saline followed by 4% paraformaldehyde (Sigma-Aldrich) in PBS, pH=7.4. Brains were then postfixed with 4%
paraformaldehyde for 24 hours at 4°C and dehydrated with 20% sucrose with 0.02% azide for 24 hours at 4°C. Dehydrated brains were frozenly cut into 40-µm coronal sections with a microtome (Leica SM 2010R).
Sections were stored at 4°C in PBS with 0.02% azide before used.
3.3 Plasmids
The shYap-2282 (shYap) and the microRNA-based RNAi vector UI4-GFP-SIBR (UI4) with human Ubiquitin C promoter constructs were generously provided by David Turner (Zhang et al., 2012). Full length of mouse Yap or enhanced green fluorescence protein (GFP) was inserted into the US2 vector with human Ubiquitin C promoter (Zhang et al., 2012). For the TRIP6-expressing construct, cDNA sequence of human TRIP6 was inserted into the pEGFP-C1 expressing vector with CMV promoter (GFP-TRIP6) (Lai et al., 2014). Short hairpin TRIP6 RNA (shTRIP6) and scrambled shRNA were inserted in pSUPER vector (Lai et al., 2014). shLacZ was inserted in pLKO vector obtained from The RNAi Consortium (TRC) shRNA Library at the Broad Institute. shPP1A
construct was purchased from Santa Cruz. For puromycin selection, anti-puromycin gene was inserted into the US2 vector (Lin et al., 2012).
3.4 Primary NSC cultures
Neurosphere cultures were prepared as previously described with modifications (Lai et al., 2014; Wang et al., 2005). Forebrains from P7
79
ICR mice were dissected and transferred into ice-cold Opti-MEM. SVZ tissues dissected from the lateral ventricle were minced and dissociated with trypsin. SVZ cells were cultured in 24-well plates (two litter of mice per experiment) in SFM (DMEM/F12 and 1% N2; Invitrogen) containing 10 ng/ml bFGF (Sigma-Aldrich), 20 ng/ml EGF (Sigma-Aldrich), 2 mg/ml Heparin (Sigma-Aldrich) and 1% antibiotics at 37°C, 5% CO2 incubator for five days. Half of the media were replaced every two days.
Electroporation was performed with 1x106 NSCs dissociated from
primary neurospheres using Nucleofector (LONZA Amaxa) with Program A-033. In Yap gain-of-function experiments, 4 µg of GFP construct was co-electroporated with 6 µg of Yap or US2 control constructs. In TRIP6 overexpression and Yap knockdown experiments, 3 µg of GFP construct and 3 µg of GFP-TRIP6 construct were co-electroporated with 4 µg of shYap or UI4 control constructs. Post-electroporated NSCs were cultured at the density of 2.5x105 cells per 6-well to form secondary (2’) NSs.
For attached cell culture, 1x105 cells were plated in 24-well plates and cultured in SFM with 1% FBS without antibiotics for 1–2 hours before transfection. For gain-of-function of Yap, cells were co-transfected with 0.25 µg of GFP and 0.35 µg of Yap or US2 control constructs. For loss-of-function of Yap, cells were co-transfected with 0.25 µg of GFP and 0.35 µg of shYap or UI4 control constructs. For overexpression of TRIP6 and knockdown of Yap, cells were co-transfected with 0.25 µg of GFP and 0.25 µg of GFP-TRIP6 or control construct plus 0.35 µg of shYap or UI4 control construct. Lipofectamine® 2000 (Invitrogen) was used for transfection according to the manufacturer’s instruction. Media were
80
replaced with SFM with 1% FBS and 1% antibiotics six hours after transfection. Transfected cells were cultured for three days on cover slips coated with poly-L-lysine (Sigma-Aldrich) and laminin (Invitrogen).
3.5 Immunofluorescence
Cells were rinsed once with phosphate-buffered saline (PBS, pH 7.4) and then fixed in 4% paraformaldehyde for 15 min. After washed with 1X PBT (PBS with 0.2% Triton X-100), cells were incubated in goat serum blocking buffer for one hour prior to incubation with the following primary antibodies in species-appropriate combinations at 4°C for 24 hours: rabbit anti-GFP (1:1000; Invitrogen), mouse anti--neuronal classⅢ β-Tubulin (Tuj1, 1:1000, Convance) or mouse anti-GFAP (1:1000,
Millipore). Labeling was visualized with DyLightTM 550- or 488-conjugated goat anti-mouse or anti-rabbit IgG secondary antibody (1:1000; Abcam) at room temperature for two hours. After wash, cell nuclei were stained by DAPI (Invitrogen) for 30 min and cells were mounted with anti-fade media (Pro-Long Gold, Invitrogen). For BrdU incorporation, 5 µM of BrdU was added into the media two hours before fixation. Cells were fixed and treated with 2N HCl at 37°C for 15 min and 0.1 M of sodium borate for 10 min at room temperature before
blocking. Cells were then incubated with rat anti-BrdU (1:500; Accurate) at 4°C for 24 hours. Labeling was visualized with DyLightTM
594-conjugated goat anti-rat IgG secondary antibody (1:1000; Abcam).
Immunofluorescence procedure for brain sections was described in Wu et al (Wu et al., 2013). Slices were incubation with the following primary
81
antibodies: rat anti-BrdU (1:500; AbD serotech), mouse anti-NeuN (1:1000; Millipore), rabbit SOX2 (1:1000; Millipore), rabbit anti-Ki67 (1:250; Millipore), or guinea pig anti-Doublecortin (1:5000;
Millipore). Labeling was visualized with DyLightTM 488-conjugated goat anti-rat, DyLightTM 488 goat anti-rabbit, DyLightTM 550 goat anti-mouse (1:500, abcam) or DyLightTM 549 donkey anti-guinea pig (1:500, Jackson ImmunoResearch).
3.6 P19 cell culture
The mouse embryonic carcinoma cell line P19 cells were cultured in MEMα (Invitrogen) with 7.5% calf serum (HyClone), 2.5% fetal bovine serum (FBS; HyClone) and 1% Penicillin-Streptomycin-Glutamine (Invitrogen) at 37°C, 5% CO2 incubator and maintained subconfluent prior to transfection (Farah et al., 2000). 6x105 cells were culture in 6-well plates. In TRIP6 gain-of-function experiments, cells were
co-transfected with 1 µg of anti-puromycin, 1.4 µg of TRIP6 or GFP control constructs. In TRIP6 loss-of-function experiments, cells were
co-transfected with 1 µg of anti-puromycin, 1.4 µg of shTRIP6, or scramble control constructs. Ten hours after transfection, cells were cultured in Opti-MEM (Invitrogen) containing 15 µg/ml puromycin (Sigma-Aldrich), 1% FBS and 1% antibiotics for one or three days.
For PP1A knockdown, 6x105 cells were cultured in 6-well plates. Cells were co-transfected with 1 µg of anti-puromycin, 1.4 µg of TRIP6 plus 1.4 µg of shPP1A or shLacZ control constructs. Ten hours after
82
transfection, cells were cultured in Opti-MEM (Invitrogen) containing 15 µg/ml puromycin, 1% FBS and 1% antibiotics for 24 hours.
To examine the subcellular localization of YAP, 1.2x105 cells were cultured in 12-well plates. Cells were transfected with Yap construct plus TRIP6 or Ctrl constructs and cultured in a low cell density in Opti-MEM (Invitrogen) containing 1% FBS and 1% antibiotics for 24 hours. After immunolabeling, fluorescence intensities of cytosol and nuclei were circled and quantified by using Image J. Transfected cells with accumulation of nuclear YAP were counted as N>C cells.
3.7 HEK293T cell culture
HEK293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% FBS and 1% Penicillin-Streptomycin-Glutamine at 37°C, 5% CO2 incubator. For co-immunoprecipitation, cells were transfected with flag-TRIP6 or flag plasmid in 10-cm dishes.
3.8 Co-immunoprecipitation
To examine protein-protein interaction between TRIP6, Lats1 or PP1A, cells were harvested in co-immunoprecipitation buffer (1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 mM HEPES, 1 mM EDTA, 1 mM EGTA) supplemented with a mixture of protease inhibitors and
phosphatase inhibitors. Lysates were sonicated for ten seconds to partially disrupt the cell membrane and centrifuged at 13,000 rpm, 4°C for 15 min.
Endogenous TRIP6 was immunoprecipitated with mouse anti-TRIP6 antibody (BD) and SureBeads™ Protein G Magnetic Beads (BIO-RAD).
83
Flag-TRIP6 was immunoprecipitated with anti-FLAG M2 monoclonal antibody-conjugated agarose (Sigma-Aldrich). Protein complex was resolved by SDS-PAGE, and transferred to the nitrocellulose membrane for immunoblotting. Proteins were detected with rabbit anti-TRIP6 (Bethyl Laboratories), anti-FLAG rabbit polyclonal antibody (Sigma-Aldrich), rabbit anti-Lats1 (Bethyl Laboratories) or rabbit anti-PP1A antibody (abcam).
3.9 Western blot analysis
Tissues or cells were lysed in SDS lysis buffer (10% SDS, 60 mM Tris-HCl pH6.8) and sonicated for 20 seconds. Cytosol and nuclear samples from transfected P19 cells were extracted with Subcellular Protein
Fractionation Kit for Cultured Cells (Thermo). The protein concentration was determined using Bio-Rad Protein Assay. Equal amount of protein extracts (20 µg) were separated by sodium dodecylsufate-poly-acryamide gel electrophoresis (SDS-PAGE) and immunoblotting was carried out according to standard methods. Primary antibodies used for Western blot analysis were mouse β-Tubulin (1:1000; Sigma-Aldrich), rabbit anti-Nucleolin (1:1000, abcam), rabbit anti-p-LATS1/2 (1:1000; Cell
Signaling), or rabbit anti-LATS1/2 antibody (1:1000; abcam). To visualize protein levels, HRP-conjugated goat rabbit or goat anti-mouse secondary antibodies (1:20000; Jackson ImmunoResearch) and ECL kit (Thermo) were used. Chemiluminescent was detected by
Luminescent image analyzer Las4000. Signal intensities of Western blot analysis were quantified by using Image J and signal intensities of
84
experimental groups were normalized to their controls at the same membrane.
3.10 Luciferase assay
For the activity of YAP, P19 cells in 12-well plates were co-transfected with 0.05 µg of US2-renilla Luciferase construct, 0.5 µg of CTGF-firefly
For the activity of YAP, P19 cells in 12-well plates were co-transfected with 0.05 µg of US2-renilla Luciferase construct, 0.5 µg of CTGF-firefly