advances.sciencemag.org/cgi/content/full/4/10/eaat2111/DC1

Supplementary Materials for

Cell chirality regulates intercellular junctions and endothelial permeability

Jie Fan, Poulomi Ray, Yao Wei Lu, Gurleen Kaur, John J. Schwarz, Leo Q. Wan*

*Corresponding author. Email: wanq@rpi.edu

Published 24 October 2018, Sci. Adv. 4, eaat2111 (2018)

DOI: 10.1126/sciadv.aat2111

The PDF file includes:

Detailed targeting sequences of shRNA plasmids Fig. S1. The hUVEC monolayer permeability, TEER, ZO-1, intercellular gap number, and size with over 30 nM Indo V treatment. Fig. S2. VE-cadherin morphology at different levels of PKC activation. Fig. S3. PKC activity, metabolic activity, motility, and expression of cadherin proteins within the dosage range of Indo V causing cell chirality reversal. Fig. S4. Chirality of endothelial cells in blood vessels. Fig. S5. Determination of cell chirality using 2D micropatterning. Fig. S6. Cell chirality of hUVECs as a function of Indo V concentration. Fig. S7. Cell chirality and permeability of hUVECs as a function of TPA concentration. Fig. S8. The CCW chirality persists for 48 hours after Indo V withdrawal. Fig. S9. Cell chirality of hUVECs with FAK inhibition. Fig. S10. PKC-mediated reversal of endothelial cell chirality persists with known vascular permeability factors. Fig. S11. Activation of PKC signaling is required for the reversal of cell chirality. Fig. S12. PKCα but not other isoforms is required for the PKC-mediated reversal of cell chirality. Fig. S13. PI3K signaling is required for the PKC-mediated reversal of cell chirality. Fig. S14. AKT1/2 kinase signaling is required for the PKC-mediated reversal of cell chirality. Fig. S15. AKT1/2 kinase is down-regulated by shRNA. Legends for movies S1 to S4

Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/4/10/eaat2111/DC1)

Movie S1 (.avi format). The hUVEC migration on a micropatterned ring (inner diameter, 200 μm; outer diameter, 500 μm).

Movie S2 (.avi format). Cell migration on edges of the ring (inner diameter, 200 μm; outer diameter, 500 μm) during 42 to 46 hours in movie S1. Movie S3 (.avi format). The hUVEC migration after TPA treatment on a micropatterned ring (inner diameter, 200 μm; outer diameter, 500 μm). Movie S4 (.avi format). TPA-treated cell migration on edges of the ring (inner diameter, 200 μm; outer diameter, 500 μm) during 46 to 58 hours in movie S3.

Detailed targeting sequences of shRNA plasmids PKC shRNA is a pool of five different shRNA plasmids with hairpin sequences:

5’-GATCCACCAAGCAGAAGACCAACATTCAAGAGATGTTGGTCTTCTGCTTGGTTTTTT-3’

5’-GATCCCACTGCACCGACTTCATCTTTCAAGAGAAGATGAAGTCGGTGCAGTGTTTTT-3’

5’-GATCCTCAGTCCATCAACAAGCAATTCAAGAGATTGCTTGTTGATGGACTGATTTTT-3’

5’-GATCCGGGATGTGCAAAGAGAACATTCAAGAGATGTTCTCTTTGCACATCCCTTTTT-3’

5’-GATCCCAGAGAAGCACGTGTTTGATTCAAGAGATCAAACACGTGCTTCTCTGTTTTT-3’ PKCα shRNA plasmid with the hairpin sequence:

5’-GATCCAAAGGCTGAGGTTGCTGATTTCAAGAGAATCAGCAACCTCAGCCTTTTTTTT-3’

PKCδ shRNA plasmid with the hairpin sequence:

5’-GATCCCCATGAGTTTATCGCCACCTTCAAGAGAGGTGGCGATAAACTCATGGTTTTT-3’ PKCε shRNA Plasmid (h) is a pool of three different shRNA plasmids with hairpin sequences:

5’-GATCCCAGAGGAATCGCCAAAGTATTCAAGAGATACTTTGGCGATTCCTCTGTTTTT-3’

5’-GATCCCTGGATGAGTTCAACTTCATTCAAGAGATGAAGTTGAACTCATCCAGTTTTT -3’

5’-GATCCGAGACCTCATGTTTCAGATTTCAAGAGAATCTGAAACATGAGGTCTCTTTTT-3’

Akt1/2 shRNA plasmid with the hairpin sequence:

5’-GATCCTGCCCTTCTACAACCAGGATTCAAGAGATCCTGGTTGTAGAAGGGCATTTTT-3’

Supplementary Figures and Captions

Fig. S1. The hUVEC monolayer permeability, TEER, ZO-1, intercellular gap number, and

size with over 30 nM Indo V treatment. (A, B) Permeability and TEER of the hUVEC

monolayer with Indo V treatment. Data are presented as average ± s.d. (n=3). “*” represents

significant difference at p<0.05. (C-E) Percentage of positively stained ZO-1 along entire cell

border, intercellular gap number, and area of Indo V treated cell monolayers on the transwell

membrane. Data are presented as average ± s.d. (n=11 images for 0 nM group, n= 12 images for

20-25 nM groups, n=13 images for 30 nM group, n=6 images for 50 and 100 nM groups). “*”

represents significant difference at p<0.05. (F) Number of hUVECs after 48 hours Indo V

treatment. Data are presented as average ± s.d. (n=12). “*” represents significant difference at

p<0.05.

50%

60%

70%

80%

90%

100%

Indo V (nM)

ZO-1 staining

Pe

rcen

tage

**

0%

2%

4%

6%

8%

10%

Indo V (nM)

Cell gap area

*

Perc

enta

ge o

f ga

p a

rea

0

500

1000

1500

2000

2500

Indo V (nM)

Cell gap numbers*

Cel

l gap

nu

mb

ers

per

mm

2

05

10152025

Indo V (nM)

Permeability

*

P alb

um

in(x

10

-6cm

/s)

0

10

20

30

Indo V (nM)

TEER

**

Elec

tric

al r

esis

tan

ce (

Ω)

D

A

E

B

C

0

200

400

600

800

1000

0 10 20 30 50 100 200

Cel

l nu

mb

er p

er m

m2

Indo V (nM)

Cell number

*

F

Fig. S2. VE-cadherin morphology at different levels of PKC activation. (A) Upper panel:

Immunofluorescence images of hUVEC monolayers on transwell membrane labeled with Alexa

Fluor 568 VE-cadherin (F8, red), and lower panel: the corresponding color-scaled fluorescence

intensity. The white dashed windows in the upper panel were twice zoomed in and shown at the

right bottom corners in the lower panel (Scale bar: 50 μm). The VE-cadherin stripes in the 20 nM

-20

0

20

40

60

80

0 1 2 3 4 5

Inte

nsi

ty

Distance (μm)

Kmean = 1.56-20

0

20

40

60

80

0 1 2 3 4 5

Inte

nsi

ty

Distance (μm)

Kmean = 1.46-20

0

20

40

60

80

0 1 2 3 4 5

Inte

nsi

ty

Distance (μm)

Kmean = 0.380

0.51

1.52

2.5

0 20 30

Kurt

osi

s

Indo V (nM)

****

Kurtosis of VE-cad intensity

50%60%70%80%90%

100%

0 20 30

Indo V (nM)

Pe

rcen

tage

VE-cad staining

A

B

G

C D E

0 nM Indo V 20 nM Indo V 30 nM Indo VV

E-ca

d

0

85

F

-50

0

50

100

-50

0

50

100

-50

0

50

100

0 0.2 0.4 0.6 0.8 1

VE-cad intensity profiles

Inte

nsi

ty

Position

Indo V treated group exhibit decreased peak intensity and increased width compared to the other

two groups (0 nM and 30 nM Indo V). (B-D) Intensity profiles along a 5 μm line perpendicular to

the VE-cadherin junction (as demonstrated by the yellow line with the 0 nM Indo V group in A)

of the cells treated with Indo V at 0 nM (B), 20 nM (C), and 30 nM (D). Data are presented as

average ± s.d. (n=206 junctions for 0 nM group, n= 205 junctions for 20 nM groups, n=220

junctions for 30 nM group). (E) Comparison of the kurtosis (peakedness) of VE-cadherin

intensity profiles in (B-D). VE-cadherin was more diffused at the cell-cell junction with 20 nM

Indo V treatment. Data are presented as average ± s.d. (n=6 images for each group). “*”

represents significant difference at p<0.05, “**” at p<0.01, and “***” at p<0.001 by One-way

ANOVA analyses with the Bonferroni-Holm method between groups. (F) The fluorescence

intensity profile along the entire border of a cell (blue, red and green circles shown in (A),

respectively. The reported intensity was calculated as the junctional VE-cadherin intensity

subtracted by average cytoplasm intensity. The positive intensity represents junctional VE-

cadherin formation. (G) Percentage of positively stained VE-cadherin pixels along cell border.

Compared with the ZO-1 result in Fig. 1F, no significant differences (One-way ANOVA) in VE-

cadherin intensity were observed within the Indo V dosage range from 0 to 30 nM. Data are

presented as average ± s.d. (n=6 images for each group).

Fig. S3. PKC activity, metabolic activity, motility, and expression of cadherin proteins

within the dosage range of Indo V causing cell chirality reversal. (A) Relative PKC activity of

the Indo V treated hUVECs quantified using ELISA. Data are presented as average ± s.d. (n=3).

(B) MTT activity of the total hUVECs with different levels of PKC activation within the

experimental culture period. Data are presented as average ± s.d. (n=3). No significant differences

0

0.02

0.04

0.06

0

10

15

20

25

30

10

0

20

0Indo V dosage (nM)

PKC activation

PKC

(n

g/μ

g to

tal p

rote

in)

***

**

*

0

2

4

6

8

10

0 20 30Indo V (nM)

Cadherin expression

N-cad VE-cad

No

rmal

ized

inte

nsi

ty

0

100

200

300

400

0 10 15 20 25 30

Net

mig

rati

on

(p

ixel

s)

Indo V (nM)

Cell motility

-200

0

200

-200 0 200

Y (p

ixel

s)

X (pixels)

Cell tracks (0 nM)

-200

0

200

-200 0 200

Y (p

ixel

s)

X (pixels)

Cell tracks (10 nM)

-200

0

200

-200 0 200

Y (p

ixel

s)X (pixels)

Cell tracks (15 nM)

-200

0

200

-200 0 200

Y (p

ixel

s)

X (pixels)

Cell tracks (20 nM)

-200

0

200

-200 0 200

Y (p

ixel

s)

X (pixels)

Cell tracks (25 nM)

-200

0

200

-200 0 200

Y (p

ixel

s)

X (pixels)

Cell tracks (30 nM)

I J

C D E

F G H

A

0

1

2

3

0 hr 24 hrs 48 hrs 72 hrs

Cellular metabolic activity 0 nM Indo V10 nM Indo V15 nM Indo V20 nM Indo V25 nM Indo V30 nM Indo V50 nM Indo V100 nM Indo V200 nM Indo V500 nM Indo V1000 nM Indo V

No

rmal

ized

MTT

act

ivit

y

*

**

B

were observed within the Indo V dosage range from 0 to 30 nM; however, significant decreases

were observed at higher doses (≥50 nM). (C-H) Normalized tracks of cell migration for 4 hours

with the Indo V treatment at 0 nM (C), 10 nM (D), 15 nM (E), 20 nM (F), 25 nM (G), and 30 nM

(H). (I) The distance of net cell migration described above. Data are presented as average ± s.d.

(n=20 for each group). No significant changes were found with the dosage range of Indo V from 0

nM to 30 nM. (J) Normalized N- and VE-cadherin expression on the confluent hUVEC

monolayer with different levels of PKC activation. Data are presented as average ± s.d. (n=6 for

each group).

Fig. S4. Chirality of endothelial cells in blood vessels. (A) Maximum intensity projection of

immunofluorescence of mouse Vena Cava showing ZO-1 (1A12, Alexa Fluor 594, red), VE-

cadherin (C19, Alexa Fluor 647, magenta), cell nuclei (DAPI, blue) and centrosomes (pericentrin,

0%20%40%60%80%

100%Biases of cell centroids

Left Neutral Right

** *** *

Tissue Left Neutral Right Total

Vena Cava(mouse)

163 142 242 547

Thoracic aorta(mouse)

294 259 350 903

Aortic arch(mouse)

89 124 140 353

Umbilical vein(human)

59 108 85 252

— Cell borders ● Centrosomes● Cell centroids ● Nuclei centroids

A B C

D E F

Vena Cava Thoracic aorta Aortic archG

VE-

cad

/ ZO

-1/

Peri

cen

trin

/ D

AP

I

Left Right Neutral Unidentifiable

VE-cad / ZO-1 /Pericentrin / DAPI

— Cell borders ● Cell centroids Nuclei ● Nuclei centroids

Alexa Fluor 488, green) (Scale bar: 100 µm). (B) Image segmentation along cell-cell junctions

(red) in (A), shown with cell centroids (yellow). (C) Extraction of cell nuclei (blue) in (A), shown

with nuclear centroids (cyan). (D) Merged image for cell chirality analysis including cell borders

(red), centrosomes (green), nuclei centroids (blue) and cell centroids (yellow). (E) Numbers of

left biased, right biased, or non-biased (neutral) endothelial cells in blood vessels, as defined in

Fig. 2D. (n=4 mice, N=547 cells for vena cava; n=3 mice, N=903 cells for thoracic aorta; n=3

mice, N=353 cells for aortic arch; n=2 human umbilical cords, N=252 cells for umbilical vein).

Bold red font indicates dominant chirality and a significant difference at p<0.05 by the rank test.

(F) Percentage of biases of endothelial cells in the blood vessels. “*” represents significant

difference at p<0.05, “**” at p<0.01, and “***” at p<0.001 by the rank test. (G) Typical

immunofluorescence images of mouse vena cava, thoracic aorta, and aortic arch, shown with ZO-

1 (1A12, Alexa Fluor 594, red), VE-cadherin (C19, Alexa Fluor 647, magenta), cell nuclei

(DAPI, blue) and centrosomes (pericentrin, Alexa Fluor 488, green). Biases of individual cells

were color-coded and shown in the lower panel (Scale bar: 100 µm).

Fig. S5. Determination of cell chirality using 2D micropatterning (A-D: control CW cells; E-

H: Indo V treated CCW cells). (A, E) Phase contrast images of micropatterned hUVECs (Scale

bars: 100 μm). (B, F) Cell alignment direction determined by an automated Matlab program based

on the intensity gradient and shown by short blue lines. (C, G) The circular histogram of biased

angles (B) and (F) shows CW (C) and CCW (G) chirality, respectively. (D, H) Circumferential

averages of the subregional biased cell alignment angles. The negative angles represent CW cell

alignment, while positive angles represent CCW cell alignment.

10%20%30%

30°

60°90°

-90° -60°

-30°

0°

10%20%30%

30°

60°90°

-90° -60°

-30°

0°

A B DC

E F HG

150 200 250 300 350 400 450 500 550-5

0

5

10

15

20

25

30

Radial position (μm)

Cir

cum

fere

nti

al

aver

age

angl

e (˚

)

150 200 250 300 350 400 450 500 550-30

-25

-20

-15

-10

-5

0

5

Radial position (μm)

Cir

cum

fere

nti

al

aver

age

angl

e (˚

)

Fig. S6. Cell chirality of hUVECs as a function of Indo V concentration. (A) Numbers of CW,

NC, and CCW rings at different Indo V concentrations. CW: clockwise alignment; NC: non-chiral

alignment; CCW: counterclockwise alignment. The bold in red font indicates dominant chirality

and a significant difference at p<0.05 by the rank test. (B) Percentage of CW, NC, CCW cell

alignment. The chirality of hUVECs switched from CW to CCW dominantly when the Indo V

dosage varied from 10 nM to 30 nM. Data in (B) are presented as average ± s.d. “*” represents

significant difference at p<0.05, “**” at p<0.01, and “***” at p<0.001.

BIndo V CW NC CCW Total

0 nM 410 36 4 450

0.1 nM 92 5 1 98

1 nM 94 4 1 99

10 nM 286 26 5 317

15 nM 140 63 21 224

20 nM 83 97 65 245

25 nM 57 68 92 217

30 nM 4 35 171 210

50 nM 1 15 143 159

100 nM 14 122 168 304

200 nM 0 28 139 167

500 nM 0 40 114 154

1000 nM 13 92 217 322

A

0%

20%

40%

60%

80%

100%

120%

Indo V (nM)

Cell chirality

CW NC CCW

****** *** *** *

* ********

**

Fig. S7. Cell chirality and permeability of hUVECs as a function of TPA concentration. (A)

Phase contrast images and cell alignment angles of the micropatterned hUVECs at different doses

of TPA (Scale bar: 100 µm). (B-D) Numbers and percentages of CW, NC and CCW rings and the

corresponding chiral factors of hUVECs with TPA treatment. The bold red font in (B) indicates

dominant chirality and a significant difference at p<0.05 by the rank test. The chirality switched

from CW to CCW dominantly when the TPA dosage varied from 0.1 nM to 0.5 nM. Data in (C,

D) are presented as average ± s.d. “*” represents significant difference at p<0.05 and “***” at

p<0.001. (E, F) Permeability and TEER of the hUVECs with the TPA treatment. Data are

presented as average ± s.d. (n=3). “**”represents significant difference at p<0.01 and “***” at

p<0.001.

-1.5-1

-0.50

0.51

1.5

0 0.1 0.2 0.3 0.5 1TPA (nM)

Chiral factor

CW

CCW

*

*0%

40%

80%

120%

0 0.1 0.2 0.3 0.5 1TPA (nM)

Cell chiralityCW NC CCW

****

*** ***

TPA dosage CW NC CCW Total

0 nM 225 66 5 296

0.1 nM 48 21 0 69

0.2 nM 18 31 14 63

0.3 nM 80 91 66 237

0.5 nM 5 40 177 222

1 nM 7 31 226 264

0 nM 0.2 nM TPA 0.3 nM TPA 0.5 nM TPA 1.0 nM TPA0.1 nM TPAA

B

30°

60°

90°-90°

-60°

-30° 0°30°

60°

90°-90°

-60°

-30° 0° 30°

60°

90°-90°

-60°

-30° 0° 30°

60°

90°-90°

-60°

-30° 0° 30°

60°

90°-90°

-60°

-30° 0°30°

60°

90°-90°

-60°

-30° 0°

02468

1012

0 0.1 0.2 0.3 0.4 0.5TPA (nM)

Permeability

*** ***

P alb

um

in(x

10

-6cm

/s)

0

5

10

15

20

25

0 0.1 0.2 0.3 0.4 0.5TPA (nM)

TEER of hUVECs

*** **

Elec

tric

al r

esis

tan

ce (

Ω)

C

E F

D

Fig. S8. The CCW chirality persists for 48 hours after Indo V withdrawal. (A) Phase contrast

images of the micropatterned hUVECs at 24~120 hours after pre-treatment with Indo V for 24

hours (Scale bar: 100 µm). (B, C) Numbers and percentages of CW, NC, and CCW ring after

drug withdrawal. The bold red font in (B) indicates dominant chirality and a significant difference

at p<0.05 by the rank test. The chirality gradually switched back from CCW to CW dominantly

after 96 hours. Data in (C) are presented as average ± s.d. “*” represents significant difference at

p<0.05 and “***” at p<0.001. (D, E) Cell number and metabolic activity of cell mixture with

different ratios. Data are presented as average ± s.d.

24 hrs afterIndo V withdrawal

48 hours afterIndo V withdrawal

72 hours afterIndo V withdrawal

96 hours afterIndo V withdrawal

120 hours afterIndo V withdrawal

0%25%50%75%

100%

24 48 72 96 120

Cell chiralityCW NC CCW

Time after Indo V withdrawal (hours)

***

*

A

C

Time afterIndo V withdrawal

CW NC CCW Total

24 hours 3 34 142 179

48 hours 25 64 82 171

72 hours 40 63 49 152

96 hours 89 51 30 170

120 hours 104 36 45 185

B

0

0.5

1

1.5

Cell mixture (CW : CCW)

Cell metabolic activity

No

rmal

ize

d M

TT

acti

vity

0200400600800

Cell mixture (CW : CCW)

Cell number

Cel

l nu

mb

er p

er m

m2

D E

Fig. S9. Cell chirality of hUVECs with FAK inhibition. (A, B) Numbers of CW, NC and CCW

rings and the corresponding chiral factors of hUVECs treated with FAK inhibitor 14 (Y15) with

or without 30 nM Indo V. The bold red font in (A) indicates dominant chirality and a significant

difference at p<0.05 by the rank test. Data in (B) are presented as average ± s.d. “**” represents

significant difference at p<0.01 and “***” at p<0.001.

-1.5-1

-0.50

0.51

1.5

Chiral factor

Ctrl Indo VCW

CCW

** *** ******

Groups CW NC CCW Total

Ctrl 133 2 0 135

Y15 (1 μM) 31 0 0 31

Y15 (2.5 μM) 74 9 5 88

Y15 (5 μM) 90 2 3 95

Indo V 13 6 64 83

Y15 (1 μM) + Indo V 8 11 70 89

Y15 (2.5 μM) + Indo V 4 17 52 73

Y15 (5 μM) + Indo V 2 21 46 69

A B

Fig. S10. PKC-mediated reversal of endothelial cell chirality persists with known vascular

permeability factors. (A, B) Numbers of CW, NC and CCW rings and the corresponding chiral

factors of hUVECs treated with VEGF, PAF or histamine under the condition of PKCα inhibition

by 1 μM Gö6976 or PKCα activation by 30 nM Indo V. The bold red font in (A) indicates

dominant chirality and a significant difference at p<0.05 by the rank test. Data in (B) are

presented as average ± s.d. “*” represents significant difference at p<0.05 and “**” at p<0.01.

-1.5

-1

-0.5

0

0.5

1

1.5

VEG

F (1

0 n

g/m

L)

VEG

F (1

00

ng/

mL)

PAF

(0.1

μM

)

PAF

(1 μ

M)

His

tam

ine

(1 μ

M)

His

tam

ine

(10

μM

)

Chiral factor

Ctrl Gö6976 Indo V

CW

CCW

* * * ** * *

A

B

Groups

VEGF(10 ng/mL)

VEGF(100 ng/mL)

PAF(0.1 μM)

PAF(1 μM)

Histamine(1 μM)

Histamine(10 μM)

Ctr

l

Gö

69

76

Ind

o V

Ctr

l

Gö

69

76

Ind

o V

Ctr

l

Gö

69

76

Ind

o V

Ctr

l

Gö

69

76

Ind

o V

Ctr

l

Gö

69

76

Ind

o V

Ctr

l

Gö

69

76

Ind

o V

CW 83 67 1 90 83 4 89 63 2 80 71 5 93 76 1 92 60 3

NC 3 21 14 2 8 17 1 23 13 1 13 15 3 8 9 1 12 16

CCW 1 1 63 2 0 53 0 5 67 0 1 71 0 3 77 1 4 69

Fig. S11. Activation of PKC signaling is required for the reversal of cell chirality. (A, B)

Western blot analysis of the PKC/PKCα phosphorylation of hUVECs treated with 30 nM Indo V

or 0.5 nM TPA. (C, D) Numbers of CW, NC, and CCW rings of hUVECs with the combined

treatment of Indo V (30 nM) / TPA (0.5 nM) and Ro-31-8425 (1 μM, a general PKC inhibitor) /

Gö6976 (0.5~1 μM, an inhibitor of conventional PKC isoform). The bold red font indicates

dominant chirality and a significant difference at p<0.05 by the rank test. “***” in (D) represents

significant difference at p<0.001.

A

C

Groups CW NC CCW Total

Ctrl 194 1 0 195

Indo V 7 30 55 92

Indo V + Ro-31-8425 73 19 3 95

Indo V + Gö6976 (0.5 μM) 71 23 4 98

Indo V + Gö6976 (1 μM) 81 9 2 92

TPA 15 17 60 92

TPA + Ro-31-8425 72 20 2 94

TPA + Gö6976 (0.5 μM) 66 17 6 89

TPA + Gö6976 (1 μM) 84 3 1 88

B

Phospho-PKC (Thr497)

β-actin

DM

SO

80 kDa

42 kDa

Ind

o V

TPA

Phospho-PKCα(Thr638)

β-actin

80 kDa

42 kDa

DM

SO

Ind

o V

TPA

-1.5-1

-0.50

0.51

1.5

Chiral factor

CW

CCW

*** ***

D

Fig. S12. PKCα but not other isoforms is required for the PKC-mediated reversal of cell

chirality. (A) The relative mRNA levels of PKC isoforms in hUVECs analyzed by quantitative

RT-PCR. Data are presented as average ± s.d. (n=3). “***” represents significant difference at

p<0.001. (B, C) Numbers of CW, NC and CCW rings and the corresponding chiral factors of

hUVECs transfected with shRNA of PKC isoforms with the treatment of Indo V (200 nM) or

TPA (10 nM). The bold red font in (B) indicates dominant chirality and a significant difference at

p<0.05 by the rank test. Data in (C) are presented as average ± s.d. “*” in (C) represents

significant difference at p<0.05.

-1.5-1

-0.50

0.51

1.5

DMSO Indo V TPA

Chiral factor

Ctrl shRNA

PKC shRNA

PKCα shRNA

PKCδ shRNA

PKCε shRNA

CW

CCW

*

*

0

0.5

1

1.5

Rel

ativ

e m

RN

A le

vel

qPCR

Ctr

l sh

RN

Ap

an-P

KC

shR

NA

PK

Cα

shR

NA

Ctr

l sh

RN

A

PK

Cδ

shR

NA

pan

-PK

C s

hR

NA

Ctr

l sh

RN

A

PK

Cε

shR

NA

pan

-PK

Csh

RN

A

PKCα PKCδ PKCε

*** *** ***

Groups CW NC CCW Total

Ctrl shRNA 193 1 0 194

PKC shRNA 181 9 3 193

PKCα shRNA 183 15 0 198

PKCδ shRNA 154 14 3 171

PKCε shRNA 125 6 0 131

Ctrl shRNA + Indo V 3 18 91 112

PKC shRNA + Indo V 86 44 12 142

PKCα shRNA + Indo V 90 25 2 117

PKCδ shRNA + Indo V 10 33 73 116

PKCε shRNA + Indo V 9 38 107 154

Ctrl shRNA + TPA 8 43 108 159

PKC shRNA + TPA 78 68 16 162

PKCα shRNA + TPA 91 82 14 187

PKCδ shRNA + TPA 18 46 38 102

PKCε shRNA + TPA 13 34 80 127

C

BA

Fig. S13. PI3K signaling is required for the PKC-mediated reversal of cell chirality. (A, B)

Numbers of CW, NC and CCW rings and the corresponding chiral factors of hUVECs with

combined treatment of Indo V (30 nM) or TPA (0.5 nM) and Wortmannin (0.5~1 μM). The bold

red font in (A) indicates dominant chirality and a significant difference at p<0.05 by the rank test.

Data in (B) are presented as average ± s.d. “*” represents significant difference at p<0.05, “**” at

p<0.01, and “***” at p<0.001.

Groups CW NC CCW Total

Ctrl 378 51 4 433

Indo V 7 30 55 92

Indo V + Wort (0.5 μM) 32 14 43 89

Indo V + Wort (1 μM) 89 4 1 94

TPA 0 45 186 231

TPA + Wort (0.5 μM) 38 65 62 165

TPA + Wort (1 μM) 73 40 7 120

A B

-1.5-1

-0.50

0.51

1.5Chiral factor

CW

CCW

***

*

**

*

Fig. S14. AKT1/2 kinase signaling is required for the PKC-mediated reversal of cell

chirality. (A) Western blot analysis of the AKT phosphorylation of hUVECs treated with 30 nM

Indo V or 0.5 nM TPA. (B, C) Phase contrast images and numbers of CW, NC and CCW of the

micropatterned hUVECs with the combined treatment of Indo V (30 nM) or TPA (0.5 nM) and

AKT1/2 kinase inhibitor (AKT1/2 Inh, 1 µM) (Scale bar: 100 µm). The bold red font in (C)

indicates dominant chirality and a significant difference at p<0.05 by the rank test. “*” in (D)

represents significant difference at p<0.05, “**” at p<0.01, and “***” at p<0.001.

A

Groups CW NC CCW Total

Ctrl 194 1 0 195

Indo V 7 30 55 92

Indo V + AKT inhibitor

62 22 14 98

TPA 15 17 60 92

TPA + AKT inhibitor

60 21 15 96

CB

Phospho-AKT (Ser473)

β-actin

60 kDa

42 kDa

DM

SO

Ind

o V

TPA

-1.5-1

-0.50

0.51

1.5Chiral factor

CW

CCW

*** **

Fig. S15. AKT1/2 kinase is down-regulated by shRNA. The relative mRNA levels of AKT1

and AKT2 in hUVECs analyzed by quantitative RT-PCR. Data are presented as average ± s.d.

(n=3). “***” represents significant difference from the control at p<0.001.

0

0.2

0.4

0.6

0.8

1

1.2

Rel

ativ

e m

RN

A le

vel

qPCR

Ctr

l sh

RN

A

AK

T1/2

sh

RN

A

Ctr

l sh

RN

A

AK

T1/2

sh

RN

A

AKT1 AKT2

***

***

Supplementary Video Captions

Movie S1. The hUVEC migration on a micropatterned ring (inner diameter, 200 μm; outer

diameter, 500 μm). After cell attachment, time-lapse images were captured every 5 min for a

total of 46 hours. The video is played at 35 fps.

Movie S2. Cell migration on edges of the ring (inner diameter, 200 μm; outer diameter, 500

μm) during 42 to 46 hours in movie S1. The video is played at 4 fps.

Movie S3. The hUVEC migration after TPA treatment on a micropatterned ring (inner

diameter, 200 μm; outer diameter, 500 μm). After cell attachment, time-lapse images were

captured every 5 min for a total of 58 hours. The video is played at 35 fps.

Movie S4. TPA-treated cell migration on edges of the ring (inner diameter, 200 μm; outer

diameter, 500 μm) during 46 to 58 hours in movie S3. The video is played at 10 fps.