Total RNA was prepared from myofibroblast cultures replated at
intermediate density. After 18 hours, the medium was changed to
DMEM/F12 medium, as indicated with either 1% or 10% FBS and with
FGF-2 (20 ng/ml), heparin (5 μg/ml), or TGF-β (1 ng/ml). We chose
to lyse the cultures after 24 hours to capture changes in mRNA that
precede the phenotypic expression of α-SM actin protein and its
incorporation into microfilaments, which we detect after 3 to 5 days
(see above). We lysed the cells in TriZol Reagent, following the
manufacturer’s instructions (Molecular Research Center, Inc.,
Cincinnati, OH). Samples were separated on a 1% agarose-formaldehyde
gel and transferred to a Nytran nylon membrane (Schleicher & Schuell,
Keene, NH). The membrane was hybridized with a
32P-labeled cDNA probe corresponding to a unique
111 nucleotide sequence of the 3′ untranslated region of rabbit α-SM
actin (nucleotides 1114–1335)
23 at 37°C for 16 to 18
hours. To normalize the RNA loading in each lane, the same filter was
hybridized in NorthernMax Hybridization Buffer (Ambion, Austin, TX)
with a
32P-labeled plasmid cDNA insert of the
internal standard, 18S ribosomal RNA (DECA templates; Ambion), and
washed using solutions provided by Ambion (NorthernMax Wash Buffer
System).
24 25 In each detection, the blot was exposed to
Biomax MS film (Eastman Kodak, Rochester, NY) and developed in an
X-Omat (Eastman Kodak). The autoradiograms were scanned with a BioRad
1650 densitometer (Hercules, CA) and quantified using Image
Quant software (Molecular Dynamics, Sunnyvale, CA).