(a) Effect of doxycycline-inducible (150 ng ml-1; 3+3
days) PML silencing (sh4, sh5) on p27 and PML protein expression (representative
of at least 3 experiments) on MDA-MB-231 cells. (b-c)
Quantification of p27 and PML protein levels along 6 days of
doxycycline-inducible PML silencing on MDA-MB-231 cells (n=3) with 2 different
shRNAs. (d) Immunofluorescence quantification of nuclear p27
positive cells upon PML inducible silencing on MDA-MB-231 cells (n=4).
(e-g) Immunofluorescence quantification of the correlation of
p27 positive cells and PML levels in these cells (e-f) and
representative images of p27 and PML staining (g) upon
doxycycline-inducible PML silencing in MDA-MB-231 cells (n=4). (h)
Effect of doxycycline-inducible (150 ng ml-1; 3+3 days) PML silencing
(sh4) on RB phosphorylation (Ser780) (representative of 3 experiments) on
MDA-MB-231 cells. (i) Expression of p27-related cell cycle genes
upon PML inducible silencing in MDA-MB-231 cells (n=3). (j) Impact
of doxycycline-inducible PML silencing (sh4) on p27 and PML protein expression
on established MDA-MB-231 xenografts. Error bars represent s.e.m. p, p-value
(*/$p< 0.05,
**/$$p< 0.01,
***/$$$p< 0.001). One-tailed
one-sample t-test (b-c, i) and one-tailed Student´s t-test were used for
cell line data analysis (d-f). sh4 and sh5: shRNA against PML,
dox: doxycycline. #: Unspecific band. Molecular weight markers (kDa) are shown
to the right.