Science - USA (2022-02-11)

used: 5′-CGGCAAGCCTTGCTGCCATG(AGG)-
3 ′and 5′-TGGACAGCGAGCAGCTGCGC(CGG)-
3 ′. sgRNAs were cloned individually into PX461
vectors [RRID:Addgene_48140 ( 36 )] and co-
transfected as a pair into wild-type MDCK cells
using Lipofectamine 2000 (Thermo Fisher
Scientific). The transfected cells were treated
with 11mM nutlin-3 for 6 days to enrich for

non-growth-inhibitedp21KOcells. The pool

was then plated at one cell per well in 96-well

plates; the resulting clones were expanded and

verified by immunofluorescence and sequenc-

ing. For use in the competition experiment, a

p21KOGFP-positive population was obtained

by infecting thep21KOG5 clone with a lentiviral

construct containing pGIPZ-GFP-NLS-Puro as

described in ( 24 )andselectedusing0.65mg/ml

puromycin.

To overexpress p21, p21 cDNA was amplified

from a cDNA library obtained from MDCK

scribbleshRNA cells treated with tetracycline

to inducescribbleknockdown, as these cells

have been shown to express high levels of p21

( 24 ). The resulting polymerase chain reaction

Kozyrskaet al.,Science 375 , eabl8876 (2022) 11 February 2022 7 of 10

H

A B

p53/p21 levels

Normal High

wound-induced

damage

CDK Inhibition

Leader fate

p53

p21 mechanical

loser fate

C D

EGF

I

0

5

10

15

Apoptotic events /cell (%)

WT p21KO

)

%(

sll

ec

(^) /
st
ne
ve
(^) c
it
ot
po
p
A
Density:High Low High Low
p=0.0006
p=0.0033
p21OE : WT
Monoculture (n=5)
Co-culture (n=6)
Hours: 0 24 48 72 96 100
0
500
1000
1500
2000
Number of p21OE cells
(% of T0)
WT (scratched)
0hrs 12hrs 26hrs 38hrs
0hrs 6hrs 10hrs 14hrs
WT
0 n=60
20
40
60
80
100
% eliminated
Surrounded
spontaneous
leaders
% eliminated
0
10
20
30
40
50
n=1769
n=171
Scratch-
induced
leaders
Non-
leaders
p<0.0001
0 20 40 60 80 100
0
200
400
600
800
1000
Co-culture (n=9)
Monoculture (n=3)
p21KO : p53KO +Nutlin
Hours:
Number of
p21KO
cells (% of T0)
WT : p21OEppppppppppp
Zo-1
DAPI
J
Fig. 6. Leader cells are eliminated upon epithelial repair.(A) Movie stills
following the fate of a spontaneous leader (arrow). (B) Percentage of
spontaneous leaders that are eliminated when surrounded by neighbors.
(C) Movie stills of scratch-induced leaders (blue, yellow, and pink arrows point
to different leaders over time), showing their elimination at gap closure.
(D) Percentage of leader or surrounding nonleader cells eliminated at gap
closure. (E) Quantification of apoptotic events (cleaved caspase-3-positive cells)
in wild-type orp21KOnutlin-3–treated cells grown at low- or high-density
and after compaction. (F) Mean number ofp21KOcells (percentage of initial
number/field) in nutlin-3–treated pure cultures (monoculture) or confronted with
p53KOcells (coculture). (G) Mean number of p21OE cells (percentage of initial
number/field) in pure cultures (monoculture) or confronted with wild-type cells
(coculture). (H) Confocal imaging of ZO-1 immunostaining of a coculture at
confluency of p21OE and GFP-positive wild-type cells. (IandJ) Diagram and model
summarizing the function of p53 and p21 in epithelial repair. Epithelial scratching
induces p53 and its target p21, which drives leader cell migration and epithelial
closure through CDK inhibition. p53 (but not p21) elevation also induces leader cells
to behave as mechanical losers, causing their elimination upon epithelial closure.
Bar charts in (B) and (D) show mean values. Black bars indicate median in (E).
Error bars indicate ±SEM in (F) and (G). Thenvalues indicate the number of cells
[(B) and (D)] or the number of fields [(F) and (G)]. Data from one representative
repeat of three biological replicates [(E) to (G)], pooled from three biological
replicates [(B) and (D)], or from one selected field of three biological replicates (H).
Pvalues from logistic regression (D) or from Mann-WhitneyUtest (E).
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