Material & Methods
Materials
Cells: Rat adrenal medullary PC12 pheochromocytoma neuronal cells, mouse N1E-115 neuroblastoma cells were
all purchased from ATCC (Manassas, VA).
Reagents: Cell culture materials including Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS)
and horse serum were purchased from Mediatech Inc. (Manassas, VA). TUJ-1 monoclonal rabbit antibody against
neuronal class III ß-tubulin was purchased from Covance Inc. (Gaithersburg, MD). Monoclonal mouse antibody
against acetylated α-Tubulin, Akt1(B-1) mouse monoclonal antibody, phosphorylated-Akt 1/2/3 (Ser 473) rabbit
polyclonal antibody, rabbit polyclonal phosphorylated-Smad1 (Ser463/Ser465) antibody, β-Actin mouse
monoclonal antibody, goat anti-rabbit HRP-coupled IgG were purchased from Santa Cruz Biotech Inc.(Santa Cruz,
CA). Rabbit polyclonal antibody against tyrosine hydroxylase (TH) was purchased from Chemicon International,
Inc. (Temecula, CA, USA). Anti-mouse IgG, HRP-linked Antibody was purchased from Cell Signaling Tech. (Danvers,
MA). Goat serum, Texas Red® Goat Anti-Rabbit IgG antibody, Alexa Fluor® 488 Goat anti-Mouse IgG antibody,
4',6-Diamidino-2-Phenylindole, Dilactate (DAPI) and AlamarBlue® were from Molecular Probes-Invitrogen (Eugene,
OR). 2.5S Nerve Growth Factors (NGF) mouse natural and human recombinant BMP-2 protein was purchased
from R&D Systems (Minneapolis, MI). Human recombinant Noggin protein, DMH-1, PP-242, Nocodazole, kainic
acid (KA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and cresyl violet dye were purchased from
Sigma-Aldrich (St. Louis, MO). Laminin, mouse was purchased from BD Bioscience (Sparks Glencoe, MD).
Potassium bisperoxopicolinatooxovanadate (bpV (pic)) was purchased from Santa Cruz Biotech Inc. (Santa Cruz,
CA). Biotinylated anti-rabbit IgG and avidin–biotin–peroxidase reagents (Elite Vectastain ABC kit) were purchased
from Vector Labs (Burlingame, CA, USA).
Apparatus: Cellular apparatus including T-75cm2 culture flasks, 6-well cell culture multiwall plate, cell culture
inserts for 6-well plate, and Neurite Outgrowth Assay Kit was purchased from Milipore (Billerica, MA). BCA
Protein Assay Kit was purchased from Pierce-Thermo/Fisher Scientific (Rockford, IL). Novex® 10-20% Tris-Glycine
mini gels were purchased from Invitrogen Inc. (Eugene, OR). Microscope (BX51) and digital microscope camera
(DP72) were purchased from Olympus (Tokyo, Japan).
Methods
Neuronal Cell Culture
Rat adrenal medullary PC12 rat pheochromocytoma neuronal cells were supplemented with 7.5% fetal bovine
serum (FBS), 7.5% horse serum (ES) and 0.5% penicillin streptomycin in T-75cm2 flasks that were maintained at
37°C in a 5% CO2 incubator. Cells were split at 50% confluence by gently mechanically detaching them from the
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flask and propagated at a split ratio 1:7. We also used mouse N1E-115 neuroblastoma cell line. This cell has 36
hours of doubling time and 1:3 of sub cultivation ratio. N1E-115 cells were grown in DMEM without sodium
pyruvate, and FBS was added at a final concentration of 10%. Rat brain cortex primary neurons were used (Lonza,
MD; cat. no. R-Cx-500) according to an established procedure [1]. Each vial thawed and resuspended in 10 ml of
growth medium according to the manufacturer’s recommendations, and 1 ml/well volume of cell suspension was
seeded onto previously poly-D lysine coated wells of 24-well plates. Medium was replenished every 3 to 4 days
and cells were used for the experiment at day 21. At day 24, cells were fixed and immunostaining was performed.
For neurite protection assay, PC12 cells were seeded to 6-well plates with seeding density of 5.0x104
cells/scaffold (empirically determined as optimal seeding density) and incubated for 24-48 hr until the cell
confluency was reached 60~70%. Then, PC12 cells were differentiated with NGF (50ng/mL) for 72-120 hr and
treated with nocodazole (1µM) for 1hr to induce neurite degeneration. After nocodazole treatment, the old
media containing nocodazole were switched with fresh media containing human recombinant BMP2 protein
(50ng/mL) or BMP2 protein plus signaling inhibitor (10 µM) and incubated for 3 additional days. Neurite status
was monitored before nocodazole treatment, after nocodazole treatment and for every 24 hrs using
photomicroscope. After 72 hr incubation, PC12 cells were either fixed for immunofluorescence assay or analyzed
using NIS-Element BR image software for total neurite quantification.
For neurite outgrowth assay, PC12 cells or N1E115 cells were seeded to 6-well plate with 1.0x105 cells/well
seeding density. After the cell confuency was reached to 60~70%, neuronal cell differentiation was initiated –
with NGF(50ng/mL) for PC12 cells and with differentiation media (DMEM, 2mM L-glutamine, 1% penicillinstreptomycin and 1mM Sodium Pyruvate) for N1E115 cells. After 24 hr of incubation, human recombinant BMP2
protein (50ng/mL) or BMP2 protein plus signaling inhibitor (10 µM) was added to the wells in 6-well plates and
incubated for two additional days. Neurite status was monitored for every 24 hrs.
PC12/Astrocyte Co-Culture
For co-culture with rat PC12 cells, we chose CRL-2005 cells, type 1 phenotype astrocytes that are derived from rat
brain. CRL-2005 cells were grown in T-75cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 0.5% penicillin streptomycin. The medium was changed
every other day and cells were split at 80% confluence at a ratio of 1:6. Since CRL-2005 cells easily grow in a
variety of media, we opted for adapting the astrocytes to grow in the PC12 cell culture medium (7.5% fetal bovine
serum (FBS), 7.5% horse serum (ES) and 0.5% penicillin streptomycin in DMEM). Astrocyte-conditioned media
(ACM) was prepared by incubating CRL-2005 cells with PC12 culture media for 24 hr.
For co-culture assay, we used 6-well plates with cell culture inserts. In neurite protection assay, PC12 cells were
seeded on the well of 6-well plate first (1.0x105cell/well) and differentiated with NGF (50ng/mL) for 72 hrs,
followed by nocodazole (1 µM) treatment for 1 hr to induce neurite degeneration. After nocodazole treatment,
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CRL-2005 astrocyte cells were seeded (1.0x105cell/well seeding density) on cell culture insert and incubated with
the PC12 cells in PC12 cell culture media for 3 additional days. Also, after nocodazole treatment, differentiated
PC12 cells were cultured with ACM or with PC12 cell culture media for 3 additional days. In neurite outgrowth
assay, PC12 cells were seeded on cell culture insert (1.0x105cell/well) and CRL-2005 cells were seeded on the
bottom of the well and cultured together with NGF (50ng/mL), or PC12 cells were cultured with ACM containing
NGF or with PC12 cell culture media containing NGF. Neurite status was monitored per each day and after 72 hr
incubation, PC12 cells were either fixed for immunofluorescence assay or used for total neurite quantification.
Cell proliferation
Cell proliferation was analyzed every second day, over a period of up to 6 days using the Alamar Blue staining
method (cite). Briefly, the cells washed once in PBS and then incubated for 3 hours with their respective growth
medium supplemented with 5% Alamar Blue reagent. Supernatants were collected and their fluorescence
intensity was evaluated by excitation at 560 nm and emission at 595 nm using an ELISA Synergy 4 reader. The
data, expressed as mean ± SD, is presented in arbitrary fluorescence units.
Neurite Quantification
For quantification of total neurites, we used Neurite Outgrowth Assay Kit (Milipore) with spectrophotometer. To
induce neurite differentiation, PC12 cells were treated with NGF (50ng/mL) for 72 hours. For N1E-115 cells, the
growth media was replaced with serum-free differentiation media (DMEM, 2mM L-glutamine, 1% penicillinstreptomycin and 1mM Sodium Pyruvate) for 24 hours. After the underside of the Milicell inserts were coated
with fresh ECM protein (10µg/mL collagen for PC12 cells and 10µg/mL laminin for N1E-115 cells) for 2 hours at
37oC; 1.0x105 neuronal cells were seeded per insert, that were placed into each well of a 24 well plate. Cells were
kept at room temperature for 15 minutes for attachment, and then a total of 700µl differentiation medium was
added per well (600µl and 100µl, below and above the membrane, respectively). Neurites were let to extend for 3
days and then the inserts were fixed with -200C methanol for 20 minutes at room temperature, followed by fresh
PBS rinse. Next, inserts were placed into 400µl neurite staining solution for 30 minutes at room temperature, and
after cell bodies were removed by a moistened cotton swab, each insert was placed onto 100µl Neurite Stain
Extraction Buffer (Millipore). Finally the solutions were transferred into a 96 well plate and quantified on a
spectrophotometer by reading absorbance at 562 nm.
Immunofluorescence
After cell culture, growth media were removed and the cells were fixed with 10% formalin at room temperature
for 15 minutes. Afterward, the cells were washed with a 0.5-M glycine solution in PBS and blocked overnight at
40oC with 5% Goat Serum and 0.2% Triton-X solution in PBS. For immunostaining with primary antibodies, cells
were incubated overnight at 40oC with TUJ-1 monoclonal rabbit antibody against neuronal class III ß-tubulin
(1:200 dilution) for total neurite staining and with monoclonal mouse antibody against acetylated α Tubulin
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(1:100 dilution) for stable neurite staining. Once cells were washed three times with 1X PBS buffer (10
minutes/wash), secondary antibodies - Texas Red® goat anti rabbit IgG (1:200 dilution) for TUJ-1 antibody and
Alexa Fluor® 488 goat anti mouse IgG (1:200 dilution) for acetylated α Tubulin antibody - were added and
incubated overnight at 40oC. Subsequently, the cells were washed three times in 1X PBS buffer (10 minutes/wash)
and 1µg/ml 4', 6-Diamidino-2-Phenylindole; Dilactate (DAPI) was added after the second washing step for staining
cell nuclei. After final washing, cells were prepared to be examined using fluorescence microscope. The excitation
and emission wavelengths are 488nm/519nm for Alexa Fluor® 488-IgG (green), and 595/615 nm for Texas Red®
goat anti rabbit IgG (red) and 405/461 nm for DAPI. Fluorescence images of the cells were acquired at different
magnifications and analyzed by “ImageJ” image processing and analysis program.
Western Blot
After cell culture, PC12 cells were collected into 1.5 mL eppendorf tubes, washed with 1X TBST buffer (1X TBS
buffer with 0.1% Tween-20) and centrifuged to prepare cell pellets. 1X RIPA buffer (with 1X protein inhibitor
cocktail + 0.1 ~ 1 mM PMSF) was added to each cell pellet for resuspension. For cell lysis, resuspended cell
mixtures were frozen by liquid nitrogen for 1 min and thawed at 37oC water bath for 5 min. After 5 times of
freeze-thaw cycle, the cell mixtures were transferred to 1 mL syringe and repeatedly sprayed through 27G needle
(3~5 times). Then, the cell mixtures were under ultracentrifugation (15,000 x g for 20 min at 4°C) and the
supernatants were collected to check their total protein concentration using BCA protein assay kit.
SDS-PAGE was performed with Novex® 10-20% Tris-Glycine gradient gel, followed by transfer process with PVDF
(polyvinylidene fluoride) membrane. After transfer, the PVDF membrane was blocked with 5% nonfat milk (in 1X
TBST buffer) for either 1 hr at room temperature or overnight at 4oC. Incubation of primary antibodies (anti
phosphorylated-Smad1 (Ser463/Ser465) antibody with 1:500 dilution or anti phosphorylated-Akt 1/2/3 (Ser 473)
antibody with 1:400 dilution or anti β-actin antibody with 1:500 dilution) were performed for 1 hr at room
temperature. After washing with 1X TBST buffer, secondary antibodies – either anti-mouse or anti-rabbit HRPconjugated IgG (1:8000 dilution) were incubated for another 1 hr at room temperature. After final washing,
manually prepared ECL (enhanced chemiluminescence) were applied to the PVDF membrane for 5~10 min for film
development.
Reference
[1] Barghorn S, Nimmrich V, Striebinger A, Krantz C, Keller P, Janson B, et al. Globular amyloid β- peptide1-42
oligomer–a homogenous and stable neuropathlological protein in Alzheimer’s disease. 2005;95:834-847
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Figure S1. BMP2 Signaling is Effective in Stabilization of Neurite Microtubule Structure. Differentiated PC12 cells
were treated with Nocodazole (0.5~1 μM) for 1 hr and incubated with fresh media containing BMP2 (50ng/mL) ±
different signaling inhibitors (10 μM/each) for 3 days. A~L) Fluorescence microscopic images from
immunofluorescence(IF) assay. Stable neurites were shown as Green signal and total neurites were shown as Red
signal. Nuclei was stained with DAPI (4',6-Diamidino-2-Phenylindole) and shown as Blue. (A-F) Images of stable
neurites with nocodazole/BMP2 (A), with nocodazole (B) and with media only (C). Images of total neurites with
nocodazole/BMP2 (D), with nocodazole (E) and with media only (F). (G-L) Images of stable neurites with
nocodazole/BMP2/DMH-1 (G), with nocodazole/BMP2/noggin (H) and nocodazole/BMP2/PP-242 (C). Images of
total neurites with nocodazole/BMP2/DMH-1 (J), with nocodazole/BMP2/noggin (K) and nocodazole/BMP2/PP242 (L) (Magnification = 40X).
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