Dynamic Changes in Intracellular ROS Levels Regulate Airway Basal ...

Cell Stem Cell, Volume 15 Supplemental Information

Dynamic Changes in Intracellular ROS Levels Regulate Airway Basal Stem Cell Homeostasis through Nrf2-Dependent Notch Signaling Manash K. Paul, Bharti Bisht, Daphne O. Darmawan, Richard Chiou, Vi L. Ha, William D. Wallace, Andrew T. Chon, Ahmed E. Hegab, Tristan Grogan, David A. Elashoff, Jackelyn A. Alva-Ornelas, and Brigitte N. Gomperts

B

C

0

F

ROShi

mABSCs ROShi

ROSlo

102 103 104 105 ITGA6 ROSlo

**

5 4 3

ROShi ROSlo

*

2 1 0

p63

K5 K14 Ngfr

K

1.6

**

***

Unsorted

a

** **

1.2 0.8 0.4

I

0

P0

P0 P1 P1 (Number of passages)

ROSlo mABSCs

P2

hABSCs ROShi

*** 6.0 4.0 2.0 0.0 P1 P0 (Number of passages)

J

ROSlo

b100

***

8.0

P3

***

***

50

0

P1 P0 (Number of passages)

ROSlo mABSCs Untreated

1 μM H2O2

0.5 mM NAC + 1 μM H2O2

1.0 mM NAC

0.1 mM NAC + 1 μM H2O2

0.1 mM NAC

0.5 mM NAC

* 100

50 0

-

5 10 100 500 Paraquat (μM) 1 μM H2O2

5.0 mM NAC

1.0 mM NAC + 1 μM H2O2

5.0 mM NAC + 1 μM H2O2

Scale:

100 μm

K5 K8 DAPI

0.1 mM NAC K5 K8 DAPI

0.1 mM NAC + 1 μM H2O2 K5 K8 DAPI

L

0.5 mM NAC + 1 μM H2O2 K5 K8 DAPI

1.0 mM NAC K5 K8 DAPI

1.0 mM NAC + 1 μM H2O2 K5 K8 DAPI

5.0 mM NAC K5 K8 DAPI

10 μm

5.0 mM NAC + 1 μM H2O2 K5 K8 DAPI

160

ROSlo mABSCs derived spheres

100

***

120 80 40

M

% Control

0.5 mM NAC K5 K8 DAPI

% Control (no. of spheres)

ROSlo mABSC derived spheres

Scale:

P2

**

150

Untreated

K5 K8 DAPI

ROSlo Mito Sox

P1

P3 Scale: 100 μm

G

Mouse ROShi DHE

P1

***

0 H2O2 (μM) 1 1 1 GSH (mM) 0 0.5 1

***

80 60 40 20 0

1 5

*** 1 10 100 DPI (nM)

*** DMSO

6

hi v/s

% control (no. of spheres)

H mABSC ROS Relative quantification

% Cells seeded (no. of spheres)

104 103 ROSlo 102 102 103 104 105 ITGA6

104 103 102

mABSCs (P3)

102 103 104 105 ITGA6

102 103 104 105 ITGA6

% Cells seeded (no. of spheres)

mABSCs

105

mABSCs (P2)

102 103 104 105 ITGA6

105

b ROS (Mito Sox)

50

mABSCs (P1)

% (EdU+/ DAPI+ nuclei)

ROSl0 ROShi ROS (DHE)

Ea

***

P1 P0 (Number of passages)

20 μm

Scale:

100

mABSCs ROShi ROSlo

ROSlo

***

ROS 102 103 104 105

% (EdU+/ DAPI+ nuclei)

ROShi

ROS 102 103 104 105

(P0) sphere K5 EdU DAPI

D ROS 102 103 104 105

Figure S1 AABSCs derived

0.1 0.5 1 APO (mM)

Figure S2 A

B

POL BrdU 1

0 hours

12

BrdU 2

24

36

BrdU 3

48

60

72

12 hpi

72 hpi

24 hpi

uninjured

Sacrifice 1 Sacrifice 2 Sacrifice 3 20 μm

48 hpi Scale: Tracheal isolation and embedding H&E and IF analysis

POL: Polidocanol

D

C

K5 K8 PIGR DAPI K8 PIGR DAPI 24 hpi

24 hpi

24 hpi

Brdu K5 CC10 DAPI Brdu K5 CC10 DAPI 24 hpi 24 hpi

Notch Brdu Tub DAPI

Brdu K5 CC10

24 hpi

K5 K8 PIgR DAPI Notch K8 Tub DAPI

48 hpi 48 hpi

Brdu K5 CC10 Brdu K8 PIgR DAPI K8 PIGR DAPI 72 hpi

72 hpi 72 hpi

Brdu K5 CC10 DAPI Brdu K5 CC10 DAPI 10 μm 72 hpi

72 hpi

N K8 Tub DAPI

Brdu K5 CC10 72 hpi

uninjured

uninjured

Brdu K5 CC10 DAPI Brdu K5 CC10 DAPI

uninjured

Scale:

Brdu K5 CC10

50 µm

K5 K8 PIgR DAPI K8 PIGR DAPI uninjured

K5 Tub BrdU DAPI

uninjured

Scale:

uninjured 10 μm CC10 K8 BrdU DAPI Scale:

uninjured

Day 14 ALI culture

Day 4 Submerged monolayer culture

F

Submerged monolayer culture mABSCs

K5 K8 DAPI Untreated

K5 Tub DAPI

K5 Tub DAPI

PIGR DAPI

PIGR DAPI

LACTOFERRIN DAPI

LACTOFERRIN DAPI

TROP2 P63 DAPI

TROP2 P63 DAPI

CC10 DAPI Scale:

10 μm

H2O2 1μM

NAC 1mM

CC10 DAPI Scale:

10 μm

50 μm

Scale:

G

H2O2 1μM + NAC 1mM 20 μm

Relative % of dividing cells (EdU+/DAPI+nuclei) as per control

E

K8 PIGR DAPI 48 hpi

48 hpi

Brdu K5 CC10 DAPI Brdu K5 CC10 DAPI 48 hpi

48 hpi

250 200

K5+ K8+

*** ***

150 100 50

0 H2O2 (μM) 1 NAC (mM) -

** *** ** *** 1

1 1

5

1 5

Figure S3 A549 cells

A

Interphase

G1

G1-S initiation

B

B

R G G1-S

Merge

B

G R Merge G2

B

R Merge G Mitosis

Fluorescence Intensity

400

***

***

p