Biology Reference
In-Depth Information
13. X-ray fi lm.
14. Western Blot Stripping Buffer, like Restore™ (Pierce).
15. Film analyzer like Gel Doc 2000 (Bio-Rad).
2.8 Statistics
Results are expressed as mean ± standard error of the mean (SEM).
Comparisons between groups are done by using paired Student's
t-
test and a one-way analysis of variance (ANOVA). Statistical sig-
nifi cance is defi ned with
p
< 0.01.
3
Methods
3.1 Human Cell
Lines, PNA Treatment,
and Culture Conditions
1. Incubate all the cell lines in humidifi ed atmosphere of 5 %
CO
2
/air at 37 °C. Human erythroleukemic K562 cells are
maintained in suspension culture using RPMI-1640 medium.
Bronchial epithelial IB3-1 cell line, derived from Cystic Fibrosis
patients, were grown in LHC-8 basal medium, supplemented
with 5 % FBS in the absence of gentamycin. Incubate human
breast cancer MCF-7 and MDA-MB-231 in D-MEM medium
supplemented with 10 % fetal bovine serum and 2 mM
L
-
Glutamine. Determine the effects on proliferation, monitor
cell growth by determining the cell number/ml using a Z1
Coulter Counter.
2. Add increasing concentrations (from 500 nM to 10
M) of
PNAs targeting miR-221 to cell cultures. Seed K562 at initial
concentration of 1 × 10
4
cells/ml; determine cell growth,
according to cell number/ml, usually after 3, 4, and 5 days of
treatment, using the Z1 Cell Counter. Seed IB3-1 and
MDA-MB-231 cells at the initial concentration of 3 × 10
4
cells/cm
2
; seed MCF-7 at the initial concentration of 1 × 10
5
cells/cm
2
and determine the cell number/ml after 3 days of
treatment. Determine Cell number/ml after trypsin treatment
by using the Z1 Cell Counter.
μ
1. Incubate, e.g., 1 × 10
5
K562 cells in the presence of increasing
concentrations of fl uorescein-labeled PNAs (Fl-PNA-a221 and
Fl-Rpep-PNA-a221) in order to investigate the uptake of
PNA-a221 and Rpep-PNA-a221 (
see
Notes 1
and
2
).
2. Perform FACS analyses to obtaining results like shown in Fig.
1
after 12 (Fig.
1a
), 24 (Fig.
1b
), and 48 (Fig.
1c
) hours of cul-
ture. Usually after 12 h of treatment, low binding of the
Fl-PNA-a221 to target K562 cells is observed. On the contrary,
Fl-Rpep-PNA-a221 will effi ciently bind to K562 cells (Fig.
1a
).
3. Analyze the uptake of Fl-PNA-a221 and Fl-Rpep-PNA-a221
by using the BioStation instrument. Representative results are
shown in Fig.
1
(D-F) and indicate the differential uptake
(fully consistent with the FACS analysis) obtained using
3.2 Uptake of
Anti-miR-221 PNAs
