Human CST abundance determines recovery from diverse forms of DNA damage and
replication stress
Feng Wang, Jason Stewart and Carolyn Price
Supplementary Data
A
B
Tet-On
35
PD
N eg
C ST-O/E
3
18
25
PD 3
APC+GFP
APC+GFP
APC+GFP
APC+GFP
35
α- HA
α- STN 1
*
APC
α- TEN 1
PD 2 0
PD 2 5
GF P
Figure S1. Western blots and fluorescence-activated cell sorting (FACS) of CST O/E
cells. (A) Western blots of CST-O/E cells showing decline in CTC1 and STN1 expression
after prolonged culture. Cells were re-sorted every 20 PD maintain high CTC1 and STN1
expression. (B) FACS analysis used to isolate and monitor cells overexpressing CTC1
(Thy1) and STN1 (GFP). The GFP and Thy1.1 (stained with APC conjugated Thy1.1
antibody) positive cells appear in the upper right quadrant. Neg., control Tet-On cells
showing no Thy1.1 or GFP expression. PD 3, CST-O/E cells 3 PD after initial sorting.
Most cells showed GFP and Thy1.1 expression indicating successful sorting. PD 20,
CST-O/E cells were resorted after 20 PD to re-isolate cells with high CTC1 and STN1
expression. PD 25, CST-O/E cells were analyzed 5 PD after re-sorting to verify that they
retained high CST and STN1.
1
B
C
R el. G-ov erhan g Am ount
N ati ve
D enatured
Ex o -
ST
Te
ST
PD 35
3 25 50
C
/E
3 25 50 35
C
t -o
n
-O
/E
Te
ST
-O
n
C
t -o
Te
PD 35
t -o
n
-O
/E
A
3 25 50
M (k b)
1.5
6.0
5.0
1.0
4.0
0.5
3.0
0
PD 35
3
Tet-on
Ex o +
25
50
2.0
C ST-O/E
M edian
tel ome re leng th
~3 .7 k b
N
ST
sh
sh
sh
NT
ST
N
1-
1-
7
7
R
es
Figure S2. The effect of CST over-expression on telomere structure. (A) Analysis of Goverhang abundance by in-gel hybridization with (TA2C3)4 probe. (B) Quantification of ingel hybridization data (mean  S.D., n=3 experiments). (C) Telomere lengths in control
and CST-OE cells lines at various PD after doxycycline addition to induce TEN1
expression.
α- Actin in
α- STN 1
1.0
0.04
Figure S3. Western blot showing knockdown of STN1 (42 kDa) in shSTN1-7 cells and
the expression of the sh-resistant Flag-STN1 allele (shSTN1-7 Res). Control cells
expressed a non-targeting shRNA (shNT). Loading control is actinin (100 kDa). Lanes 13 were loaded with 25 μg protein. Numbers below the gel indicate the level of STN1
relative to the shNT control after normalization to actinin for loading.
2
Links to reagents
Actinin antibody:
http://www.scbt.com/datasheet-17829-alpha-actinin-h-2-antibody.html
FLAG M2 antibody
http://www.sigmaaldrich.com/catalog/product/sigma/f1804?lang=en&region=US
Histone S3 antibody
http://www.cellsignal.com/products/primary-antibodies/9715
HRP-conjugated secondary antibody
http://www.piercenet.com/product/poly-hrp-secondary-antibodies
FLAG® M2 Affinity Gel
http://www.sigmaaldrich.com/catalog/product/sigma/a2220?lang=en&region=US
PCNA antibody
(PC-10, Santa Cruz)
http://www.scbt.com/datasheet-56-pcna-pc10-antibody.html
Click-iT® EdU Alexa Fluor® 488 Imaging Kit
https://www.lifetechnologies.com/order/catalog/product/C10337
IdU antibody
https://www.bdbiosciences.com/ptProduct.jsp?prodId=22693
CIdU antibody
http://www.abdserotec.com/brdu-antibody-bu1-75-icr1-obt0030g.html
IdU
http://www.sigmaaldrich.com/catalog/search?interface=All&term=5-Iodo-2%E2%80%B2deoxyuridine&N=0&focus=product&lang=en&region=US
CIdU
http://www.sigmaaldrich.com/catalog/search?interface=All&term=5-Chloro2%E2%80%B2-deoxyuridine&N=0&focus=product&lang=en&region=US
3