Antisera and Reagents
A mouse monoclonal antibody against NF-ATc (clone 7A6, [Timmerman et al., 1997
]) was purchased from Affinity Bioreagents, (Golden, CO). A mouse monoclonal antibody against NF-ATp (clone G1-D10, [Timmerman et al., 1997
]) and a rabbit polyclonal antibody against NF-AT4/c3/x (Ho et al., 1995
) were obtained from Dr. Gerald Crabtree. For immunoblots, a rabbit polyclonal antibody against NF-ATp was purchased from Upstate Biotechnology (Lake Placid, NY). Mouse ascites and normal rabbit serum were purchased from Sigma Biosciences (St. Louis, MO). Secondary antibodies were obtained from Jackson Immunoresearch Laboratories
, (West Grove, PA). Tyramide-green was purchased from NEN Dupont (Boston, MA). SJL mice were purchased from The Jackson Laboratory (Bar Harbor, ME), whereas C3H/HEN mice were purchased from Harlan Sprague Dawley (Indianapolis, IN). NF-ATp null mice (Hodge et al., 1996
) were a gift of Dr. Laurie Glimcher. Expression plasmids for NF-ATp (pSH210) (Ho et al., 1995
), NF-ATc (pSH107c) (Northrop et al., 1994
), and NF-AT 4/x/c3 (pSH250A) (Ho et al., 1995
) were obtained from Dr. Gerald Crabtree. Except where noted, all cell culture supplies were purchased from Life Technologies (Grand Island, NY). FBS was obtained from Atlanta Biologicals (Norcross, GA). Basic fibroblast growth factor (bFGF) was purchased from Promega (Madison, WI). Calf skin collagen was purchased from Sigma. [methyl-3
H]Thymidine was purchased from Amersham (Arlington Heights, IL). Amphotropic (ATCC CRL-11554) retroviral producer cells were obtained from the American Type and Culture Collection (Rockville, MD). d
-Luciferin was purchased from Molecular Probes (Eugene, OR). Thapsigargin was purchased from LC Chemicals (San Diego, CA). Phorbol myristate acetate and ionomycin were purchased from Sigma. CSA was a gift of Sandoz (Basel, Switzerland).
Primary cultures were derived from the tibialis anterior of adult SJL/J or NF-ATp null mice 2 d after induced muscle damage, and myoblasts were purified to >99% (Rando and Blau, 1994
). Mouse myoblasts were grown in growth media (GM: Ham’s F10, 20% FBS, 5 ng/ml bFGF) on collagen-coated dishes in a humidified 5% CO2
atmosphere at 37°C. Dishes were coated with a solution of 0.01% calf skin collagen in 0.1 N acetic acid, allowed to stand overnight, and rinsed with sterile PBS. Differentiation was induced by switching confluent myoblast cultures to fusion medium (FM: DMEM, 2 or 15% horse serum) for 44–48 h. Primary human myoblasts were derived from a muscle biopsy taken from a 2-y-old donor and were purified to >99% using flow cytometry (Webster et al., 1988
). Human myoblasts were grown in Ham’s F10, 15% FBS, 5 ng/ml bFGF on collagen-coated dishes in a humidified 5% CO2
atmosphere at 37°C. Differentiation of human myoblasts was induced by switching confluent myoblasts cultures to FM containing either 2 or 5% horse serum for 48–80 h.
Cell Proliferation Assays
Human myoblasts (2 × 104) were plated in 24-well collagen-coated tissue culture plates in muscle growth medium. At 24–72 h after plating, the cells were pulsed with 1 μCi/ml [methyl-3H]thymidine (specific activity, 25 Ci/mmol) for 2 h at 37°C. DNA synthesis was assayed by measuring the amount of radioactivity incorporated into trichloroacetic acid-insoluble material. The cells were washed twice with cold PBS, treated with cold 20% trichloroacetic acid for 30 min at 37°C, and lysed for 10 min with 0.1 N NaOH and 0.1% SDS. The lysates were mixed with Scintisafe Econo1 scintillation fluid (Fisher, Pittsburgh, PA) and counted in a Beckmann-LS6000IC liquid scintillation counter. Triplicate wells were used to analyze each experimental condition.
Embryonic Myosin Heavy Expression
Human myoblasts were plated in six-well collagen-coated culture plates in muscle growth medium and grown to high density. High density myoblasts were preincubated in either vehicle or CSA for 24 h before switching to FM, and drug treatment was continued for another 48–80 h. Treatment time was determined by the length of time necessary to obtain extensive myotube formation in the vehicle-treated samples. This time varied depending on the lot number and percentage of serum. Duplicate wells for each condition were lysed in ice-cold RIPA-2 (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS containing protease inhibitors [90 μg/ml phenylmethylsulfonylfluoride, 0.1 mM leupeptin, 0.2 trypsin inhibitor unit/ml aprotinin, 5 μg/ml pepstatin A, 0.2 mM sodium orthovanadate, and 50 μg/ml soybean trypsin inhibitor]). Insoluble material was pelleted by centrifugation.
Equal amounts of protein (10 μg) (Bradford, 1976
) were loaded onto SDS-polyacrylamide minigels consisting of a 7.5% separating gel with a 4% stacking gel (Laemmli, 1970
) and electrophoresed at 150 V for 70 min. Proteins were transferred to an Immobilon membrane (Millipore, Bedford, MA) using a Transblot apparatus (Bio-Rad
, Richmond,CA) at 250 mA for 1 h as per the manufacturer’s protocol. After nonspecific binding was blocked by incubation of the membrane with 5% nonfat dry milk in Tris-buffered saline (TBS), the membrane was incubated overnight at 4°C with a mouse monoclonal antibody against embryonic myosin heavy chain (EyMHC) (Cho et al., 1994
). The EMyHC antibody (F.1652) was used as an undiluted hybridoma supernatant. The membranes were washed in TBS containing 0.1% Tween 20 and further incubated with a 1:10,000 dilution of a peroxidase-conjugated anti-mouse IgG in TBS containing 0.1% Tween 20 and 5% horse serum for 1 h at room temperature. After further washing, the membrane was incubated with an ECL Western blotting detection kit (Amersham) as per the manufacturer’s directions, exposed to Hyperfilm-ECL x-ray film (Amersham), and developed using an automatic film processor. Densitometry of the films was performed using an optical scanner and NIH Image software.
Creatine Kinase Assays
SJL mouse myoblasts were plated at 1.5 × 105
cells per well in 12-well plates in GM. After 4 h, vehicle or CSA was added, and 12 h later, the GM was replaced with Insulin-Transferrin-Selenium FM (DMEM, 1:100 dilution of Insulin-Transferrin-Selenium Supplement [GIBCO BRL], 0.1% BSA, 200 U/ml penicillin G, 200 μg/ml streptomycin). Cells were lysed and sonicated 24 h later in 0.05 M glycylglycine (pH 7.5), 1% NP-40, 0.1% β-mercaptoethanol. Creatine kinase (CK) activity was determined using Sigma Diagnostics procedure number DG147-UV and standardized to protein levels (Bradford, 1976
). Background, defined as nondifferentiation-specific CK activity in myoblasts maintained in GM, was subtracted from values determined in drug-treated samples (mean = 14.5% of vehicle CK activity). Values are reported as percent of vehicle CK activity (mU/mg protein). Triplicate samples were analyzed for each concentration of CSA in three independent experiments.
Induced Regeneration of Skeletal Muscle and Histological Analyses
C3H/HEN mice were anesthetized with an i.p. injection of a cocktail of 35 mg/kg ketamine and 5 mg/kg xylazine, and an incision of ~3 mm was made overlying the tibialis anterior muscles. Muscle damage was induced by direct application of a small piece of dry ice to the surface of the exposed muscle for 5 s. Groups of two to three animals received either PBS or 45 mg/kg CSA i.p daily and were killed 10 d after damage. The muscles were removed, embedded in OCT mounting medium, and frozen in isopentane cooled in liquid nitrogen. Serial cross-sections were collected onto gelatin-coated slides at 400- to 500-μm intervals along the entire length of the muscle and analyzed histologically by hematoxylin and eosin staining.
Retroviral Reporter Plasmids
The retroviral NF-AT–responsive plasmid (pKA7) contains a luciferase coding sequence under the control of a minimal human IL-2 promoter with an upstream triplex of the distal IL-2 gene NF-AT response element (Northrop et al., 1993
). The control plasmid (pKA9) is missing the NF-AT response elements, but contains the minimal IL-2 promoter. Both plasmids confer neomycin resistance and were created by modifications (Boss et al., 1998
) to the retroviral plasmid pLNCX (Miller et al., 1993
Retroviral Production and Infection
Retroviruses were prepared by transient transfection of helper-virus free amphotropic producer cells (Pear et al., 1993
) with the plasmids pKA7 or pKA9. The Bing-CAK8 producer cells were grown in producer growth media (PGM) consisting of DMEM, 4.5 μg/ml glucose, 10% FBS in a humidified 5% CO2
atmosphere at 37°C. To produce infectious retroviral supernatants, cultured cells at 50–80% confluence in 100-mm diameter dishes were transfected with the retroviral plasmids using CaPO4
and 25 μM chloroquine for 6–12 h before refeeding with 20 ml PGM. Twenty hours after the start of the transfections, the PGM was aspirated and replaced with 9 ml fresh PGM before the dishes were placed in a humidified 5% CO2
atmosphere at 32°C to enhance the retroviral titer (Kotani et al., 1994
). Supernatants containing infectious retroviruses were collected at 48, 60, and 72 h after transfection refeeding with 9 ml of PGM each time, filtered through a 0.45-μm syringe tip cellulose acetate filter, aliquoted, snap frozen in liquid nitrogen, and stored at −80°C.
SJL myoblasts in multiwell plates were infected by adding retroviral supernatant supplemented with 10% FBS, 5 ng/ml bFGF, and 4 μg/ml polybrene and spinning at 2500 rpm for 30 min at 32°C in a Beckman model GS-6R centrifuge in a swinging bucket rotor (Springer and Blau, 1997
). The retroviral supernatant was aspirated, and the cells were refed with GM and returned to the incubator. The infection protocol was repeated once more 8–12 h later. Forty-eight hours after the last infection, the cells were fed with GM containing 50 μg/ml G418. After 3–5 d of selection, the surviving cells were expanded and used for experiments through approximately 10 more passages. The efficiency of the two rounds of retroviral infections was estimated at >90% as little cell death occurred during drug selection.
Drug Treatments and Luciferase Assays
Retrovirally infected SJL myoblasts were plated at 4–7 × 104 cells per well of 24-well collagen-coated plates and used in assays as indicated. Drugs were added to muscle cultures and incubated for 6–7 h at 37°C. The cells were washed with PBS, and 75 μl of lysis buffer (1% Triton X-100, 4 mM EGTA, 25 mM Tris/phosphate, 10% glycerol, 2 mM dithiothreitol) were added to each well and incubated for 10 min at room temperature. The cell lysates were centrifuged at 13,100 × g for 5 min at room temperature, and the supernatants were removed. Luciferase assays were performed by combining 50 μl of supernatant, 350 μl of assay buffer (25 mM Tris/phosphate, 40 mM MgSO4, 4 mM EGTA, 2 mM ATP, 1 mM dithiothreitol), and 100 μl of 0.75 mM d-luciferin. Light output was measured after a 5-s delay over a 10-s window using a Turner TD-20e luminometer (Turner Designs, Sunnyvale, CA).
Immunoblotting of NF-AT Proteins
SJL mouse muscle cells were lysed in RIPA-2 containing protease inhibitors (Complete Mini, Boehringer-Mannheim, Indianapolis, IN). Cellular proteins at 100 μg/lane for NF-ATc and NF-AT4/x/c3 analyses and 50 μg/lane for NF-ATp analysis were separated using SDS-PAGE. Immunoblots were processed as described for F.1652 detection except a peroxidase-conjugated anti-rabbit IgG was used for detection of NF-ATp and NF-AT4/x/c3. Specificity of the antibodies for NF-AT isoforms in immunoblots of muscle proteins was determined by preabsorbing the individual antibodies with extracts from HEK cells transfected with expression plasmids for NF-ATc(pSH107c), NF-AT4/x/c3 (pSH250A), or NF-ATp (pSH210) or control HEK extracts.
Human myoblasts were plated at approximately 30% confluency in GM. Myoblasts were assayed at high density (80–90% confluency). To induce the formation of multinucleated myotubes, myoblasts were grown to near confluence and switched to FM. Myotubes were assayed at two stages: nascent (24 h in FM) and mature (75–80 h in FM). Cultures were treated with vehicle (0.01% DMSO) or thapsigargin (10 nM) for 10 min at 37°C. In some experiments, CSA (1 μM) was added 10 min before the addition of thapsigargin.
Immediately after the drug treatments, the cells were fixed in −20°C methanol for 5 min. To block nonspecific protein binding, the cells were first incubated in blocking buffer (PBS, 2% horse serum, 0.5% Triton X-100) for 30 min. All antibodies were diluted in this blocking solution. The cells were further incubated with either a 1:1000 dilution of the anti-NF-ATc monoclonal antibody, a 1:500 dilution of the anti-NF-ATp monoclonal antibody, or a 1:1500 dilution of the anti-NF-AT4/x/c3 antibody for 1 h. After three washes, the cells were incubated with a 1:1000 dilution of either biotinylated anti-mouse IgG (for NF-ATc and NF-ATp antibodies) or biotinylated anti-rabbit IgG (for NF-AT4/x/c3) for 30 min. After washing, antibody binding was detected using TSA-Green according to the manufacturer’s directions, but with a 1:200 dilution of the streptavidin-conjugated horseradish peroxidase. Specific staining was tested by replacing the primary monoclonal antibodies with normal ascites or with normal rabbit serum in the case of the polyclonal NF-AT4/x/c3 antibody or by omitting the primary antibody. Further specificity was demonstrated by preabsorbing the NF-ATc and NF-AT4/x/c3 antibodies with extracts from HEK cells transfected with expression plasmids for these isoforms or control HEK extracts. Specificity of the NF-ATp antibody was shown by staining of muscle cultures derived from NF-ATp null mice. All analyses and photography were performed on a Axiovert microscope (Carl Zeiss, Thornwood, NY) equipped with a video camera (Optronics Engineering, Goleta, CA) and Scion Image software (Scion, Frederick, MD).