Download Supplementary Figure Legends

Supplementary Figure Legends
Supplementary Figure S1. Disabling RB bypasses PML-induced senescence in normal human
fibroblasts. A, Immunoblots for the indicated proteins in IMR90 cells co-infected with a
retroviral vector expressing PML-IV (PML) and either a vector control (V) or derivatives
expressing CDK4 or CDK6. Samples were obtained from cell populations ten days after
infection. B, Proliferation marker KI-67 qPCR from samples of cells as in A. C, Senescenceassociatedb–galactosidase assay (SA-b-Gal) in cells expressing either a vector control (V) or
PML and a vector control (V) or derivatives expressing CDK4 or CDK6. D, Summary of qPCR
data for E2F targets gene expression showing cell cycle genes and DNA repair genes from
samples of cells as in (A). Expression levels were encoded in a color gradient that helps to
visualize the degree of derepression according to the intensity of the green color. E, QPCR for
RB family members (RB, p107 and p130) in cells expressing PML and the indicated shRNAs. F,
Knockdown of RB was sufficient to reduce PML-induced senescence and restore cell
proliferation. Data shows percent of SA-b-Gal positive cells in cells expressing PML and the
indicated shRNAs. ShNTC is a non-targeted shRNA control. G, Proliferation marker KI-67
qPCR from samples of cells as in (F). H, Summary of qPCR data for E2F targets gene expression
showing cell cycle and DNA repair genes in samples as in (F). Expression levels were encoded in
a color gradient that helps to visualize the degree of derepression according to the intensity of the
green color.
Supplementary figure S2. Cyclin-dependent kinase inhibitors p21 and p16INK4A cooperate with
PML to inhibit cancer cell proliferation. A-B, QPCR for p16INK4a (A) and p21 (B)
overexpressions. C-D, Relative growth of U2OS cells (C) and PC3 cells (D) infected with empty
vector (V) or a vector expressing PML as well as a vector expressing either p21 or p16INK4A.
Supplementary Figure S3. CDK inhibitors enhance PML-mediated growth arrest and senescence in
tumor cell lines. A-B, Relative growth of U2OS cells infected with empty vector or a vector
expressing PML treated with flavopiridol (Fla) (A) or palbociclib (Pal) (B) at the indicated doses
for six days. Error bars: mean +/- SD *=p<0.05, **=p<0.01 and ***=p<0.005 using Student ttest.
C, Fluorescent imaging of LC3B and p62 forming foci, as markers of autophagy vesicles
(red) in U2OS cells expressing empty vector (V) or a vector expressing PML and treated with
120 nM flavopiridol (Fla), 500nM palbociclib (Pal) or vehicle (Ctrl). Immunofluorescence
staining for PML is shown in green and nuclear DNA (DAPI) in blue. Scale bar= 10 μM. D-E,
Quantification of LC3B and p62 foci per cells of immunofluorescence performed in (C). Cells
presenting more than 100 foci where considered positive for induction of autophagy. Three sets
of 100 cells were looked at. Error bars: mean +/- SD, *=p<0.05, **=p<0.01 and ***=p<0.005
using Student t-test.
Supplementary Figure S4. Gene Set Enrichment Analysis (GSEA) showing homology to our gene
expression profiling. GSEA of microarray data comparing PML-expressing PC3 cells with PC3
cells bearing a vector control (A-F) or PC3 cells treated with palbociclib vs. PC3 cells treated
with vehicle (G-J). Sets showing significant enrichment with FDR values smaller than 0.11 are
shown. Comparing control cells with PML expressing PC3 cells revealed upregulation of
interferon target genes (A and B) while genes involved in the cell cycle were downregulated (C).
Many of the PML-downregulated genes overlapped with E2F3 targets (D) as expected from the
known effects of PML on E2F-targets gene expression. PML inhibited genes in the EGFR
signaling pathway (E) and triggered a quiescence gene expression pattern (F), which is consistent
with the induction of a reversible cell cycle arrest. Comparing PC3 cells treated with palbociclib
with PC3 cells treated with vehicle revealed a downregulation of cell cycle genes (G), E2F3
response genes (H) and EGFR targets (I). Palbociclib also increased the expression of genes in
the interferon gamma signaling reactome (J). Since PML or palbociclib were unable to trigger a
stable senescence in this tumor cell line, these changes are obviously not sufficient to trigger
bona fide senescence.
Supplementary Figure S5. Gene Set Enrichment Analysis (GSEA) showing homology to our gene
expression profiling. A-L, GSEA of microarray data comparing PML-expressing PC3 cells
treated with palbociclib with PC3 expressing PML or treated with palbociclib. Sets showing
significant enrichment with FDR values close to 0 are shown. Interestingly, genes involved in
chromosome maintenance were downregulated in cells treated with PML and palbociclib
(Supplementary Fig. 5G and H), while genes involved in steroid biosynthesis (Supplementary Fig.
5I and J) and olfactory receptors (Supplementary Fig. 5K and L) were enriched in
PML/palbociclib treated samples suggesting novel pathways that regulate senescence.
Supplementary Figure S6. Palbociclib or 5-Aza-dC sensitise cells to PML-induced irreversible
growth arrest. A-B, Activation of PML-ER in normal human fibroblasts induces an irreversible
senescent cell cycle arrest after three days of induction. C-F, The data set corresponds to figure 6
but includes additional controls with the ER empty fusion vector (V-ER) to discard the effects of
OHT. C-D, Schematic of the experiment timeline and results. PC3 cells expressing a V-ER or
PML-ER fusion are first treated (left) with palbociclib (Pal-100: 100 nM or Pal-500: 500 nM,
every three days for six days) or (right) 5-Aza-dC (A-120: 120 nM or A-250: 250 nM, once over
three days and then kept in normal medium for another three days). Cells are then treated with
ethanol 0.1% (CTRL: control) or 4-hydroxytamoxifen (OHT: 100 nM) every three days over the
next six days without further palbociclib or 5-Aza-dC. Growth was estimated by a crystal violet
retention assay. Results are shown as relative growth compared to cells expressing V-ER with
only control treatments. E-F, As in (C-D) but the initial palbociclib or 5-Aza-dC temporary
treatment is followed by a temporary treatment with control (CTRL: ethanol 0.1%) or 4hydroxytamoxifen (OHT: 100 nM) every three days over the next six days prior to plating for
growth assay without any further treatment. Cells were fixed in glutaraldehyde and crystal violet
retention was quantified. Results are shown as relative growth compared to cells expressing VER
with only control treatments.
Supplementary Figure S7. palbociclib decreased DNMT1 levels in a dose dependent manner.
A, Fluorescent imaging of DNMT1 (red) in cells PC3 cells infected with empty vector (V) or a
vector expressing PML treated with vehicle (Ctrl) or either 100 nM or 500 nM palbociclib (Pal100 and Pal-500 respectively). PML staining is shown in green and nuclear DNA (DAPI) in blue.
Scale bar= 10 μM. B, Results of mass spectrometry (MS) for DNMT1 in vitro phosphorylation.
A schematic representation of DNMT1 sequence is presented with yellow highlights showing
peptide sequences covered by the analysis at least twice and green highlights pointing to amino
acids with various modifications found at least once. Please note that region in a dashed orange
box indicate the region where DNMT1 isoform 1 and 2 differ of 16 amino acids (aa) changing the
position of certain aa when referring to modifications The serine in a gray circle shows Ser170
(Ser154 as called by other authors) for which immunoblots are presented in figure 7 and which
was not covered in the MS analysis. Serines in a red circle represent phosphorylated serines
identified in the MS analysis with the MS spectrum presented under the protein sequence. Of
note, CDK consensus phosphorylation sequence is “S/T P X K/R” with the first serine or
threonine being the phosphorylation target. We thus underlined the aa following the identified
phosphorylated serines. Ser127 and Ser970 (or 954) follow the consensus site. Scale bar = 10
mM.