Pokaż cały numer - Farmaceutyczny Przegląd Naukowy

Farm Przegl Nauk, 2009,2
Fig. 3. Western immunoblot analysis for prolidase in control human
dermal fibroblasts (C) and the cells submitted to oxidative stress
by treatment with 30 µM t-butylhydroperoxide (t-BHP) as described
in method section. Samples used for electrophoresis consisted of
20 µg of protein of pooled cell extracts (n = 6). Detection of β-actin
was carried out in order to provide the loading control. The arrows
indicate the molecular mass of standards. The intensity of the bands
was quantified by densitometric analysis.
Fig. 4. Western immunoblot analysis for β 1 -integrin receptor (A) and
FAK (B) in control human dermal fibroblasts (C) and the cells submitted
to oxidative stress by treatment with 30 µM t-butylhydroperoxide
(t-BHP) as described in methods section. Samples used for electrophoresis
consisted of 20 µg of protein of pooled cell extracts (n = 6).
Detection of β-actin was carried out in order to provide the loading
control. The arrows indicate the molecular mass of standards. The
intensity of the bands was quantified by densitometric analysis.
lasts were maintained in DMEM supplemented with 10% fetal
bovine serum (FBS), 2 mM glutamine, 50 U/ml penicillin, 50
µg/ml streptomycin at 37 0 C in a 5 % CO 2 incubator. Cells were
counted in a hemocytometer and cultured at 1 x 10 5 cells per
well in 2 ml of growth medium in 6 well plates (Costar). Cells
reached about 80% of confluence at day 3 and such cells were
used for assays. Cells were used in the 8th to 14th passages.
Induction of oxidative stress in human dermal fibroblasts.
Subconfluent cells were submitted to 2 repetitive oxidative
stress by treatment for 2 days with 30 µM t-butylhydroperoxide
(t-BHP) for 1 h per day according to the method
described by Dumont et al. [27]. Induction of oxidative stress
was performed in the following manner: 10 µl of 3 mM t-
BHP in DPBS was added to each well, containing 1 ml of
DMEM with 10% FBS and plates were incubated in 37 0 C
for 1 hour. After this time, medium containing t-BHP was removed,
cells were rinsed twice with DMEM and maintained
in DMEM containing 10% FBS. The next 1hour treatment
of the fibroblasts with t-BHP was performed after 24 hours,
30
Fig. 5. Western immunoblot analysis for IGF-I receptor in control
human dermal fibroblasts (C) and the cells submitted to oxidative
stress by treatment with 30 µM t-butylhydroperoxide (t-BHP) as described
in methods section. Samples used for electrophoresis consisted
of 20 µg of protein of pooled cell extracts (n = 6). Detection of
β-actin was carried out in order to provide the loading control. The
arrows indicate the molecular mass of standards. The intensity of the
bands was quantified by densitometric analysis.
Fig. 6. Time course experiment for DNA biosynthesis (measured
by [ 3 H]thymidine incorporation assay) in control human dermal fibroblasts
and the cells submitted to oxidative stress with 30 µM tbutylhydroperoxide
(t-BHP) as described in methods section. Mean
values from three independent experiments done in duplicates are
presented.
in order to let the cells recover from the previous one. Control
cultures were incubated similarly but in the absence of t-BHP.
In order to prepare the cell extracts for assays the fibroblasts
were harvested 24h after the last scheduled stress.
Determination of β- galactosidase activity. The activity
of β- galactosidase was determined according to the method
described by Chateriee et al. [28], modified by Zwierz et al.
[29]. 150 µl of 20 mM solution of substrate (p-nitrophenylβ-D-galactopyranoside
dissolved in Mc Ilvane’s phosphate-
citrate buffer pH 4.7) was mixed with 200 µl of Mc Ilvane’s
phosphate- citrate buffer pH 4.7 and 50 µl of cell extract.
After 30 minutes of incubation in 37 0 C, the reaction was terminated
by adding 1ml borate buffer pH 9.8. The amount of
released p-nitrophenol was determined colorimetrically by
absorbance at 410 nm and calculated from calibration curve
for p-nitrophenol standards. Protein concentration was
measured by the method of Lowry et al.[30]. Enzyme activity
was reported as nanomoles of released p-nitrophenol
during one minute per milligram of supernatant protein.
DNA synthesis. Cells were counted in a hemocytometer
and cultured at 1 x 10 5 cells per well in 1 ml of growth