Handling of mice was according to university guidelines and the animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan Normal University (Approval Number 101026). Adult and pregnant CD1 mice at gestation day 15.5 and 16.5 (E15.5, E16.5) were exposed to photoperiod (12L: 12D) in IVC system with unlimited food and water. The sample size of each group of experiments was at least three.
3.2 Plasmids
For the TRIP6-expressing construct, cDNA sequence of human TRIP6 was inserted into the pEGFP-C1 expressing vector with CMV promoter (Clontech, Palo Alto). Short hairpin TRIP6 RNA (shTRIP6) and
scrambled shRNA were inserted in pSUPER and pLVTHM vector (Lai et al., 2005; Lai et al., 2007; Lin et al., 2013). Enhanced green fluorescence protein (GFP), renilla Luciferase, and the human Notch intracellular domain (NICD) were inserted into the US2 vector with human Ubiquitin C promoter. Luciferase reporter constructs CBF1-WT and CBF1-MT (4xwtCBFLuc and 4xmtCBFLuc), Gli-WT and Gli-MT (8x3’Gli-BS and 8xm3’Gli-BS), TCF/LEF binding site and mutant sites (8xTOP and 8xFOP), and the human NICD (US2-NICD), Gli2 expression construct (pcDNA3.1-HisB-Gli2), and β-catenin expression construct (CAGEYFP-CAG-ctnnb1) were described previously (Lin et al., 2012).
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3.3 Fixation and sectioning
Brain fixation and sectioning was described previously (Wu et al., 2013).
In brief, adult mice were deeply anesthetized with Avertin and perfused with saline and 4% paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO). Brains were then postfixed with 4% PFA, cryoprotected, and frozenly cut into 40-mm coronal sections with a microtome (Leica). For embryonic brains, mother mice were anesthetized and perfused as
described above. Embryonic brains were frozen and cut into 30-mm coronal sections with a cryostat (Leica, Exton, PA). Three embryonic forebrain sections (120 mm apart/section) from each animal were selected for staining. Six equivalent SVZ sections (160 mm apart/section) from each adult animal throughout the anterior to posterior part were selected after the corpus callosum and before the anterior commissure were connected from both sides of the brain.
3.4 RT-PCR
Brain tissues of female pregnant mice and their fetuses at E16.5 were subjected to RNA extraction using Trizol® (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Two micrograms of total RNA were reverse transcribed by GoScriptTM kit (Promega, Madison, WI) and then 2 ml of cDNA was used for PCR reaction by GoTaq® Green Master Mix (Promega). Mouse TRIP6 forward primer:
GAAGCCCAGTGGAGGTGCTG-30. Reverse primer: 50-ACTCCAGAAGGTCCCTCCGG-30.
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3.5 NIH3T3 cell culture
NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) with 10% calf serum (Invitrogen). Two
micrograms of pLVTHM-scamble shRNA or pLVTHM-shTRIP6 were transfected into 60 to 80% confluent 3T3 cells in 6-wells using
Lipofectamine® 2000 (Invitrogen) according to the manufacturer’s
manual. Sixteen hours after transfection, cells were starved by 0.1% BSA containing medium overnight and focal adhesions were induced by 10%
FBS DMEM for 15 min (Xu et al., 2004). Cells were then fixed by 3%
formaldehyde (Bionovas, Bremerton, WA) for immunofluorescence.
3.6 Immunoblotting
Brain tissues of female pregnant mice and their fetuses at E15.5 were lysed with 1xSDS lysis buffer (10% SDS, 60 mM Tris-HCl pH 6.8) and sonicated for 20 sec. Sixty micrograms of lysates were then applied to SDS-PAGE analysis and TRIP6 (1:2,000, Bethyl Laboratories) and GAPDH (1:5,000, GeneTex) were detected by their specific antibodies.
Immunoblotting for 3T3 cells, 4 µg plasmids were used for transfection.
Two days after transfection, cells were lysed by 1xSDS lysis buffer and 40 µg of cell lysates were then used for SDS-PAGE analysis. TRIP6 (1:2,000, Clone 16, BD Bioscience, Franklin Lakes, NJ) and β-actin (1:2,000, Sigma-Aldrich) were detected by their specific antibodies.
3.7 Immunofluorescence
Immunofluorescence for 3T3 cells was described previously (Xu et al.,
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2004). In brief, fixed cells were permeablized with 0.2% Triton X-100 and blocked with phosphate-buffered saline containing 2% BSA. TRIP6 was detected by its specific antibody (1:250, Clone 16, BD Bioscience) and DyLightTM-549 goat anti-mouse antibody (1:500, Jackson
ImmunoResearch, West Grove, PA). Cell nuclei were stained by DAPI (Invitrogen). Cells were photographed by the inverted fluorescence microscope (Leica DMI3000, Exton, PA).
The immunostaining procedure on brain slices was described in Wu et al. (2013). Brain sections were incubated in blocking buffer (10% goat serum in TBS buffer) for 1 hr followed by incubation with primary
antibodies overnight at 4°C. After washing, sections were incubated with fluorescence-conjugated secondary antibodies for 2 hr and with DAPI (Invitrogen) for 30 min. Stained sections were then mounted with anti-fade solution (Invitrogen) and photographed by a confocal microscope (TCS SP2, Leica) in the Image Core at NTNU. Antibodies used: mouse anti-TRIP6 (1:250; BD Bioscience), rabbit anti-SOX2 (1:1,000;
Millipore), rabbit anti-S100β (1:1,000; Millipore), rabbit anti-Ki67 (1:250; Leica), guinea pig DCX (1:5,000; Millipore), rabbit GFAP (1:350; Sigma), rabbit MAP2 (1:1,000; Millipore), rabbit anti-Iba1 (1:1,000; Wako), DyLightTM 488 goat-anti-mouse, DyLightTM549 goat-anti-rabbit (1:500; Thermo Scientific), and DyLightTM549 donkey-anti-guinea pig (Jackson ImmunoResearch). Cells in the SVZ area were counted in 2-mm confocal sections.
Differentiated NSCs were fixed in 4% PFA for 15 min. After wash and blocking, cells were incubated overnight at 4°C with the following
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primary antibodies: mouse anti-neuronal class III β-tubulin (Tuj1,
1:1,000, Covance, Princeton, NJ), mouse anti-GFAP (1:1,000, Millipore, Billerica, MA), and rabbit anti-GFP (1:1,000, Invitrogen). Labeling was visualized with DyLightTM-550 goat anti-mouse and DyLightTM-488 goat anti-rabbit secondary antibody (1:1,000, Abcam, Cambridge, MA). Cell nuclei were stained by DAPI (Invitrogen). Cells were photographed by an inverted fluorescence microscope in the Image Core at NTNU.
3.8 Neurosphere culture and electroporation
Neurosphere (NS) cultures were prepared as previously described (Wang et al., 2005) with modifications. P7 CD1 mice were sacrificed by cervical dislocation. SVZ tissue dissected from 2-mm-thick coronal brain sections was minced and dissociated with trypsin (Sigma), hyaluronidase (Sigma), and Kynurenic acid (Sigma). SVZ cells were cultured in a 24-well dish (1.5 mice per well) with Dulbecco’s modified Eagle’s medium
(DMEM/F12) (Invitrogen) with 1% N2 (Invitrogen), 10 ng/ml bFGF (Sigma), 20 ng/ml EGF (Sigma), 2 mg/ml Heparin (Sigma), and 1 % Penicillin-Streptomycin-Gluatmax (Invitrogen) at 37°C, 5 % CO2
incubator for 5 days. Half of the medium were replaced every 2 days. For each experiment, SVZ explants from two litters of P7 mice were
combined and cultured to form primary (1’) NS first. These 1’ NS were then dissociated and transfected with different constructs for either
secondary (2’) NS formation, proliferation or differentiation experiments.
Electroporation was performed with 1x106 NSCs dissociated from 1’ NS using Nucleofector (LONZA Amaxa) with Program A-033. In TRIP6
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loss-of-function experiments, 4 µg of GFP plasmid was co-electroporated with 6 µg of shTRIP6 or scrambled shRNA plasmid. In TRIP6 gain-of-function experiments, 4 µg of GFP plasmid was co-electroporated with 6 µg of pEGFP-TRIP6 or pEGFP-C1 control plasmid. Post-electroporated NSCs were cultured at 2.5x105 cells per 6-well to form 2’ NS.
3.9 Transfection and Differentiation of NSCs
1x105 NSCs dissociated from 1’ NS were plated in 24-well dishes and cultured in DMEM/F12 with 1% N2 and 1% FBS without antibiotics for 1–2 hr before transfection. In TRIP6 loss-of-function experiments, cells were co-transfected with 0.25 µg of US2-GFP and 0.35 µg of shTRIP6 or scrambled shRNA-harboring plasmid. In TRIP6 gain-of-function
experiments, cells were co-transfected with 0.25 µg of US2-GFP and 0.35 µg of GFP-TRIP6 or GFP-expressing vector. Lipofectamine® 2000 (Invitrogen) was used for transfection according to the manufacturer’s instruction. The medium was replaced by DMEM/F12 with 1% N2, 1%
FBS, and 1% antibiotics 6 hr after transfection. For neuronal
differentiation, NSCs were cultured for 3 days on cover slips coated with poly-L-lysine (Sigma) and laminin (Invitrogen).
3.10 Luciferase assay
P19 cells were maintained in MEMα medium with 7.5% calf serum, 2.5%
FBS, and 1% antibiotics (Lin et al., 2012). For transfection, cells were plated in 12-well dishes at 80–90% confluence in MEMα with 10%
serum without antibiotics. Lipofectamine® 2000 (Invitrogen) was used for
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transfection according to the manufacturer’s instruction. P19 cells were co-transfected with 0.05 µg of US2-renilla Luciferase, 0.5 µg of pEGFP-C1, pGFP-TRIP6, US2-NICD, Gli2, or ctnnb1, and 0.7 µg of Firefly Luciferase reporter constructs with either wild type (WT) or mutated (MT) transcription factor binding sites. Six hours after transfection, medium was replaced with Opti-MEM with 1% FBS and antibiotics. SH-SY5Y cells were maintained in DMEM/F12 medium with 10% FBS, and 1% antibiotics. For transfection, 5x105 SH-SY5Y cells were plated in 12-well dishes and cultured in DMEM/F12 with 10% FBS without
antibiotics for one hour before transfection. The transfection protocol was the same as P19 cells. Six hours after transfection, medium was replaced with DMEM/F12 with 1% FBS and antibiotics. Reporter
activity was measured 24 hr after transfection using Dual-Luciferase Assay System (Promega). For normalization, firefly Luciferase activity was first normalized to renilla Luciferase activity of each group. The obtained value from the wild-type binding sites (WT CBF1) was then normalized to that of the MT CBF1. Finally, the normalized value from the control vector group was set as 100% and the value from the TRIP6 group was shown as the percentage of the control group. Samples of each experiment were plated in duplicates and each experiment was repeated for three times (n=3).
3.11 Statistical Analysis
Two-group comparisons were analyzed by two-tailed Student’s t-test.
Difference of two distributions was analyzed by Wilcoxons rank-sum
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test. All data were presented as mean ± standard error of the mean (SEM). Significant level is p<0.05.
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