Additional file 2 - Cell Communication and Signaling

Report
Additional File 2
A
B
C
200
150
-
+
-
+
Dox
MMP9
MMP2
% MMP2 Level
#14
% MMP9 Level
Neg
150
100
100
50
0
0
Neg
D
Neg
#14
Neg
#14
F
#14
-
+
Dox
150 kDa
90 kDa
G
120
100
#14
60
40
20
120
100
80
60
40
20
0
Neg
% uPA Activity
Neg
80
0
H
120
100
80
60
40
20
0
% 90 kDa Level
+
% 150 kDa level
-
% Cath. B Activity
#14
E
Neg
50
#14
120
100
80
60
40
20
0
Neg
WT14
#14
Additional Figure 2. INPP4B expression does not suppress basal levels of secreted
and cellular proteases. (A-C) PC-3 Tet-On clone #14 and negative for INPP4B clone were
cultured for 2 days  0.5 µg/ml doxycycline in serum-containing media, followed by
incubation for 24 hours without doxycycline in serum-free media as described in Materials
and Methods. Conditioned media were collected, concentrated by centrifugation, and
analyzed by gelatin zymography (A). Enzyme levels were determined by gelatin digestion in
0.1% gelatin 10% PAGE. Levels of MMP-9 (B) and MMP-2 (C) expression were quantified
by densitometry of corresponding bands and normalized to no-doxycycline control for each
clone. Bars are means ± SEM determined from 3 independent experiments. (D) Cells were
cultured as described in (A) and casein zymography was performed for analysis of caseincleaving proteases. Expression levels of the ~150-kDa band (E) and ~90-kDa band (F) were
quantified by densitometry analysis of the corresponding bands and normalized to nodoxycycline control for each clone. (G) Cathepsin B activity was assayed as described in
Materials and Methods, from 10 µg of cell lysate prepared from cells cultured as described
in (A). Data from 6 independent experiments were averaged and normalized to untreated
cells for each clone (100%). (H) uPA activity was assayed from concentrated media
harvested from cells cultured as described in (A) and Materials and Methods. Data from 4
independent experiments were averaged and normalized to untreated cells for each clone
(100%). For all graphs, open bars denote untreated cells and closed bars denote
doxycycline treated cells.

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