Science - USA (2022-02-11)

Cell culture and plasmids
MDCK cells were maintained in Dulbecco’s
minimum essential medium (21885, Thermo
Fisher Scientific) supplemented with 10% fetal
bovine serum (FBS; P30-3305, FBS Standard,
South America, PAN Biotech) in a humidified
incubator at 37°C and 5% CO 2. Wild-type MDCK
cells were obtained from Y. Fujita (Kyoto Univer-

sity); MDCK wild-type GFP-NLS cells have

been previously described ( 24 ).

MDCKp53KOGFP-NLS clones 6 and 14 were

generated by infecting a MDCKp53KOpool ( 24 )

with lentiviral construct pGIPZ-turboGFP-NLS-

Puro ( 24 ). Selection after infection was carried

out in 0.65mg/ml puromycin. The resulting

GFP-NLS–labeled pool was then plated at one

cell per well in 96-well plates. Clones were ex-

panded and verified by immunofluorescence,

sequencing, and Western blotting.

MDCKp21KOclones G5 and E9 were gen-

erated using Cas9 D10A CRISPR technology.

Single guide RNAs (sgRNAs) against canine

CDKN1Awere designed manually following

published methods ( 36 ). Target sequences

Kozyrskaet al.,Science 375 , eabl8876 (2022) 11 February 2022 6 of 10

0hrs 8hrs

WT (scratched)

Leaders Non-Leaders

0

5

10

15

20

G1 G1 to S S/G2 G1 G1 to SS/G2

Hours in phase

p=0.0005

p=0.004

A B

p53

D E

Nuclear GFP signal Nuclear GFP signal

100 μm 0 65535

p21 Reporter (scratched)

+p38 inhibitor

H I J

0hrs 12hrs 0hrs 12hrs

Scratched monolayer

No GSE-22 +GSE-22

confluent

cells in a 96wp

well

405nm laser

no laser

cells cells

scratch-wound

Hoechst

pre-treatment

and wounding

L N

p=0.0002

Speed (μm/h)

WT

n=5

p21KO

n=13

0

10

20

30

40 p=0.0002

K

F G

0

2000

4000

6000

p=0.1238p= 0.0007p=0.0045p=0.0137

T0.5h T2.5h T4.5h T6.5h

BulkEdgeBulkEdgeBulkEdgeBulkEdge

0

20

40

60

80

100

100

200

Non-

leaders

n=4471

Scratch

leaders

n=65

Nuclear p53 signal

C p<0.0001

0

10

20

30

Speed (μm/h)

+Irr

n=13

+GSE-22

p=0.8012

p=0.0120

p=0.0100

Not-Irr

n=23

NoGSE-22

+Irr

n=15

NoGSE-22

Scratched monolayer

NoGSE-22 NoIrrNoGSE-22 +Irr+GSE-22 +Irr

M

12hrs 12hrs 12hrs

Scratched monolayer

100 μm 0 65535

p21 Reporter (scratched)

0

2000

4000

6000

T0.5h T2.5h T4.5h T6.5h

p=0.9951p= 0.8852p=0.9695p=0.1778

BulkEdgeBulkEdgeBulkEdgeBulkEdge

+p38 inhibitor

Speed (μm/h)

NoGSE-22 +GSE-22

n=49 n=66

0

10

20

30

Fig. 5. Injury-induced p53 elevation drives collective migration in
epithelial repair.(A) Quantification of cell cycle phase duration in FUCCI-reporter
expressing scratch-induced leaders. (B) Movie stills showing emergence of
scratch-induced leaders (arrow) from a monolayer of wild-type cells and p53
immunostaining of the indicated field (yellow dashed lines). (C) Quantification of
nuclear p53 intensity of scratch-induced leaders and surrounding nonleaders
from experiments, as in (B). (DandE) Movie still of cells expressing p21
promoter-driven nuclear GFP after scratching (D) and corresponding quantifica-
tions (E). Images are pseudo-colored to reflect signal intensity. Edge cells are
within white dotted lines; bulk cells are within the yellow dashed lines. T indicates
time elapsed since scratching. (FandG) Movie still of cells expressing p21
promoter-driven nuclear GFP after scratching, as in (D), but in the presence of
p38 inhibitor SB202190, and corresponding quantifications (G). (HtoJ) Movie

stills of uninduced control (H) or cells expressing the p53 inhibitor GSE-22 (I)

after scratch and corresponding quantifications of migration speed (J).

(K) Quantification of migration speed of wild-type orp21KOcells after scratch.

(L) Experimental design to induce localized p53 activation at the edge of

scratched monolayers. (MandN) Movie stills of control uninduced and

nonirradiated (left panel), uninduced and irradiated (middle panel), or GSE-22Ð

expressing and irradiated (right panel) cells after scratching (M) and

corresponding quantifications of migration speeds (N). Black bars indicate

median in (A), (C), (E), (G), (J) to (K), and (N). Thenvalues indicate the number

of cells (C) or the number of movies [(J), (K), and (N)]]. Data from one

representative repeat of three biological replicates [(A), (J), (K), and (N)] or

one representative movie of three biological replicates [(E) and (G)].Pvalues

from Mann-WhitneyUtest.

RESEARCH | RESEARCH ARTICLE