cultured in vascular cell basal medium (ATCC,
#PCS-100-030) supplemented with endothelial
growth factors (Endothelial Cell Growth Kit-
VEGF, ATCC, #PCS-100-041). HUVECs were
used for experimentation at P3 to P5 after
being received.
Generation of senescent and nonsenescent MEFs
For H3K27Ac-ChIP-seq experiments, two or
three independent MEF lines were generated
and induced to senesce via irradiation (IR), serial
passaging (REP) or KRASG12V-overexpression
(OI). For identification of IR-induced SASEs,
we established the following three MEF cultures
from each independent MEF line: proliferat-
ing P3 MEFs (to derive C1 MEFs); P6 MEFs ex-
posed to 10 Gyg-radiation (^137 Caesium source)
and cultured for 2 days (to derive C2 MEFs);
and P6 MEFs exposed to 10 Gyg-radiation and
cultured for 10 days (to derive IR-senescent
MEFs). For identification of REP-induced
SASEs, we prepared two MEF cultures from
each independent MEF line: proliferating P3
MEFs (to derive C1 MEFs) and P10 MEFs cul-
tured at 20% oxygen between P4 and P10 (to
derive REP-senescent MEFs). To identify SASEs
in OI-induced senescent MEFs, cells were in-
fected with aKRASG12V-containing lentivirus
(prepared using thepLenti-PGK-ER-KRASG12V
from Addgene #35635), selected with 250mg/ml
of hygromycin B (EMD Millipore, #400052) and
then harvested (to derive C1 MEFs) or treated
with 200 nM 4-hydroxytamoxifen (4′-OHT,
1:50,000 from stock in ethanol, Sigma H7904)
to induce KRASG12Vfor 2 days (to derive C2
MEFs) or 10 days (to derive OI-induced senes-
cent MEFs). IR-, REP- and OI-induced senes-
cent MEFs were enriched by sterile FACS aspreviously described using a BD FACSAria
4-laser digital flow cytometer with FACSDiva
v8.0.1 software with 488 nm laser ( 42 ). Sorted
cells were pelleted, resuspended in fresh culture
medium, counted, and used for ChIP-seq and
RNA extraction. Small amounts of the sorted
cells were reseeded to assess the proportion of
cell that was senescent. Samples with ~70% or
more SNCs were used for H3K27ac-ChIP-seq
experiments. C1 and C2 MEFs cultures were
also subjected to FACS but here fractions
devoid of SNCs were collected. For all other
experiments involving REP-induced SNCs,
SNCs were prepared as described above. FACS-
enriched SNCs were cultured for at least
24 hours before further use. OI-induced senes-
cent MEFs were also prepared as described
above, but instead of the lentiviral KRASG12V
expression system we used MEFs derived fromSturmlechneret al.,Science 374 , eabb3420 (2021) 29 October 2021 9 of 15
Fig. 8. p21 places cells
under immunosurveillance
to establish a timer mecha-
nism that controls cell
fate.(A) (Top) Schematic
representation of the
transgenes for dox-inducible
repression of Myc-Flag–
tagged p21 (iL-p21for
inducibleLoxP/Stop/LoxP-
p21) and monitoring of
ectopic p21 expression status
via fluorescent marker
proteins (Ai139). Blue triangles
denote LoxP sites. (Bottom)
Schematic of the experimental
design with fluorescent
markers for transgenic p21
expression and repression
indicated. (B) Rates of p21
overexpression (p21+) among
hepatocytes that were positive
for Tom and eGFP (enhanced
green fluorescent protein)
in the absence of dox (p21-OE
“ON”)oronlyTomafter
dox administration (p21-OE
“OFF”). (C) Representative
image of a p21-OE hepatocyte
surrounded by three macro-
phages (arrowheads), and
quantification of fluorescent
hepatocytes joined by
≥3 F4/80+macrophages.
(D) Assessment of fluorescent
hepatocytes associated with
≥1 iNOS+cells. (E)Asin
(D) but assessing cells with
≥1 CD3e+cells. (F) Represent-
ative image of a 6dON+2dOFF
dying hepatocyte and quantification of death rates among fluorescent hepatocytes. Scale bars: 10mm [(C) and (F)]. Mice used were on a C57BL/6 pure genetic
background. Data represent means ± SEM. Experiments in the above panels were performed once. ns, not significant. P< 0.05; P< 0.01; P< 0.001. Two-way
ANOVA with Sidak’s correction [(B) to (F)].
AAi139;iL-p21
Ai139Tom+ GFP+;p21-OEONhepatocytes
Tom+ GFP+hepatocytes+ Adeno-CrepTRE2 p21-Myc-FlagWPRE iL-p21pTRE2 eGFP WPRE Ins pCAG tdTomato P2AtTA2 WPREAi139+ DoxycyclineTom+;p21-OEOFFhepatocytes
Tom+ hepatocytesBDSTOPSTOP STOPFluorescent hepatocytes
joined by1 iNOS+
cells (%)Fluorescent hepatocytes
joined by1 CD3+cells (%)Fluorescentdying hepatocytes (%)E FeGFPTomDNAFluorescent p21+
hepatocytes (%)2dON 2dON
+2dOFFns ns***6dON
+2dOFF6dON***Ai139 (n=4)
Ai139;iL-p21(n=4)Fluorescent hepatocytes
joined by3 F4/80+
cells (%)C2dON 4dON 2dON
+2dOFF6dON
+2dOFF***ns4dON
+2dOFFns6dON*** ******TomF4/80DNAeGFP*6dON
+2dOFF6dON***6dON 6dON
+2dOFF6dON 6dON
+2dOFF*****
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