Cell culture
A mouse neuroblastoma cell line, Nauro2A cells (IFO50081) were obtained from the JCRB Cell Bank (Osaka, Japan). The cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) FBS and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA) in a humidified atmosphere containing 5% CO2 at 37 ° C.
GPR137 KO neuro2A cell generation and GPR137 genetic rescue
Experimental protocols were approved by the DNA experiment safety committee of Saitama Medical University. GPR137 KO neuro2A cells were generated using the Guide-it™ CRISPR/Cas9 systems (Takara Bio Inc., Shiga, Japan). GPR137-specific gRNAs (No.1 Forward: 5′-CCGGCTCTGGCCGACGCTTCGCCT-3′ Reverse: 5′-AAACAGGCGAAGCGTCGGCCAGAG-3′; protospacer adjacent motif (PAM) sequence; TGG: No.2 Forward: 5′-CCGGAGGCATCTAGCCGGCTCCGA-3′ Reverse: 5′-AAACTCGGAGCCGGCTAGATGCCT-3′; PAM sequence; GGG) were designed using CRISPR direct [25] and synthetic oligos were ligated into Guide-it-ZsGreen1 vector. The plasmid vectors were transfected into neuro2A cells with Lipofectamine 3000 (Invitrogen). Neuro2A cells expressing ZsGreen were selected and cultured as single cells by limited dilution. A Guide-it genotype confirmation kit (Takara Bio Inc.) was used to identify the homozygous mutants. In-del detection and cloning of targeting sites were performed using a Guide-it Indel Identification kit (Takara Bio Inc.). The colonies for KO were identified by the changes in their DNA sequences.
Rescue cells were then constructed to re-express GPR137 in GPR137 KO neuro2A (KO R) cells. The full open reading frame of murine GPR137 complementary DNA (cDNA) was obtained by PCR with Pfu DNA polymerase (Promega, Madison, WI) from a cDNA library synthesized from murine mRNA using oligonucleotide primers (Forward: 5′-GAGGAAGAAGCCTCCCAATC-3′ Reverse: 5′-CACCTGGGAGAAGAGCAGAG-3′). The PCR product was then ligated into pEF6/V5-His vector (Invitrogen). The rescue plasmid vectors were subsequently transfected into GPR137 KO neuro2A cells with lipofectamine (Invitrogen). KO R cells stably expressing GPR137 were selected and cultured as single cells by limited dilution.
RNA extraction and reverse transcription (RT)-PCR
Total RNA was extracted from cells using ISOGEN (Nippon Gene, Tokyo, Japan) following the manufacturer's instructions. Total RNA was reverse-transcribed using a PrimeScript RT reagent kit (Takara Bio Inc.). The following primer sequences were used for RT-PCR: GPR137 (NO. 1 Forward: 5'-TGCTTCTGTATGGGCACAAG-3' and Reverse: 5'-CCCTATAGCAGCTGCCTGAC-3', No. 2 Forward: 5'-ATGCCAGCCGGGCCTGTTAC-3' and Reverse: 5'-AGCAGATCACGTCTGTGGTG-3').
Cell growth assay
Microculture tetrazolium technique (MTT) assay provides a quantitative measure of the number of viable cells by determining the amount of formazan crystals produced by metabolically active cells. Cells (1 × 105 cells/well) in 24-well plates were treated and 50 µl of MTT reagent [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] (FUJIFILM, Osaka, Japan) (5 mg/ml in PBS) was added to each well. The plates were incubated in a humidified atmosphere of 5% of CO2 at 37°C for 4 h. After removing the medium, formazan crystals were dissolved in 200 µl isopropanol /HCl (100 : 0.34), and the absorbance was measured using a micro plate reader (Bio-Tek, Redmond, WA) at 570 nm relative to 630 nm.
Measurement of neurite outgrowth
Cells (3× 105 cells/well) were seeded in 6-well plates and incubated for 24 h. The medium was then replaced by serum-free fresh medium with or without retinoic acid (10 nM, RA, FUJIFILM). Cells were incubated for 24h and photographed at 10 × magnification, and images were captures using a KEYENCE BZ-X710 microscope (Keyence Corporation, Osaka, Japan). Differentiated cells were defined as cells with neurites longer than twice the cell body diameter [26].
Western Blotting
Cells were homogenized on ice in RIPA buffer [50 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% DOC] containing a protease inhibitor cocktail (Calbiochem, San Diego, CA) (1 : 1000 dilution) with a tissue homogenizer (Brinkmann Instruments, Westbury, NY). Protein concentrations were determined using a BCA protein assay kit (Nacalai Tesque, Tokyo, Japan). Proteins (10 µg /lane) in lysates were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad, Redmond, WA). After blocking with 5% skim milk (MEGMILK SNOW BRAND Co Ltd, Tokyo, Japan) in PBS containing 0.05% Tween 20 (polyoxyethylene sorbitan monolaurate, Nacalai Tesque) (PBS-T), the membranes were incubated with primary antibodies overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Beverly, MA) and then washing thrice with PBS-T. The membranes were then incubated with chemiluminescence reagent (Chemi-Lumi One Super, Nacalai Tesque; ImmunoStar LD, FUJIFILM). Images of the membranes were captured using a C-DiGit blot scanner (LI-COR, Lincoln, NE) and subjected to ImageJ analysis. Each membrane was probed with anti-GAPDH antibody (1 : 1000, ABS16, Millipore, Billerica, MA), and the bands were used as loading controls. A pre-stained molecular weight marker was used to confirm expected sizes of the target proteins.
The primary antibody is anti-GPR137 (1 : 1000, 11929-1-Ab, Proteintech, Chicago, MA), anti-Cyclin D1 (1 : 1000, ab134175, abcam, Cambridge, MA), anti-PROX1 (1 : 1000, ab199359, abcam), anti-NeuroD1 (1 : 1000, ab213725, abcam), anti-STAT3 (1: 1000, MAB1799, R&B Systems, Minneapolis, MN), anti-GAP43 (1 : 1000, ab9674, abcam), anti-CREB (1 : 1000, ab32515, abcam) anti-CREB1 (1 : 1000, ab9674, abcam), anti-AKT (1 : 1000, #587F11, Cell Signaling Technology), anti-p-AKT (1 : 1000, #9271S, Cell Signaling Technology), anti-ERK (1 : 1000, #9102, Cell Signaling Technology) dilution, anti-p-ERK (1 : 1000, sc-7383, Santa Cruz, Dallas, TX).
Statistics
Multiple comparisons were performed by one-way ANOVA followed by Newman-Keuls post-hoc test or two-way ANOVA followed by post-hoc Tukey test. All data were analyzed using Graph Pad Prism Ver. 5.01 (Graph Pad Software, Inc., San Diego, CA) and expressed as mean ± SEM. p values < 0.05 were considered statistically significant.