n g e. ) Figure S2. Atg5 sh. Bcl2. + +. +. +. - +. -. + d. Lentivirus ..... Muscle injury Skeletal muscle injury was performed by injection of barium chloride (BaCl2) into.
Stem Cell Reports, Volume 11
Supplemental Information
The AMPK/p27Kip1 Axis Regulates Autophagy/Apoptosis Decisions in Aged Skeletal Muscle Stem Cells James P. White, Andrew N. Billin, Milton E. Campbell, Alan J. Russell, Kim M. Huffman, and William E. Kraus
Figure S1
12hr time point 24hr time point
a
48hr time point
0
hours in culture
48
12 hour chloroquine treatment
isolation via FACS
b
d
c
Young
MA
PI
Annexin V
Old
Geriatric
Figure S2
c
At g5
b
sc r sh
Atg5 mRNA (fold change)
a
LC3
I II
Tubulin
d
Lentivirus
Atg5 sh Bcl2
+ -
+ +
+ -
+ +
1
7K ip
Tubulin
pp2
p27
PK
p p27(Thr198)
pA M
AMPK O ld C R
d lib
d
phospho/total ratio (fold change)
p AMPK(Thr172) A
A lib
b g
-
ld
un
AICAR -
O
Yo
a
es 4 8 hly ho Is o 4 8 urs la t e ho in d ur cu s l in tu r cu e ltu re
Fr
Figure S3
+
p AMPK(Thr172) AMPK
p p27(Thr198) p27 Tubulin geriatric
Co n
a Lentivirus Time in culture (hrs)
IP: GFP
-
Wt p2 K 7 ip1 p2 K 7 ip
1(1 98 A)
Figure S4
+ +
+
24 24 48 24
p p27(Thr198)
p27Kip1(198D) sh ATG5
-
+ -
PARP total cleaved
p p27(Thr198) Eluant
b
Tubulin
p27 tubulin
Fr es 48 hly ho Iso 48 urs lat ho in ed ur cu s l in tur cu e ltu re
young
c Compound C
-
-
+
p AMPK(Thr172) AMPK p p27(Thr198) p27 Tubulin young
geriatric
+ +
Figure S5
a
Dapi
p27Kip1
Actin
Merge
Young
G
Yo
un g
Geriatric + AICAR
G er er ia ia tri tr ic c + A IC A R
Geriatric
b
Dapi
p27Kip1
Actin
Merge
p27Kip1(198A)
98 D ) (1
p2 7K ip 1
p2 7K ip 1
(1
98 A )
p27Kip1(198D)
Figure S6
G
O I er d ia tr ic
Old
Yo un g
Young
Geriatric
FIGURE LEGENDS Figure S1, related to Figure 1. Autophagy in the MuSC increases through 48 hours in culture. A. Experimental design for quantification of young MuSC LC3B punta at 12, 24 and 48 hours in culture. All cells were treated with chloroquine for 12 hours prior to collection. B. Average number of LC3B puncta per MuSC throughout the 48-hour culture time course. (N=3 independent experiments) C. Quantified cleaved PARP expression (corresponding to Figure 1C). (N=4 independent experiments) D. Annexin V x PI FACS plots of young, middle-aged, old and geriatric MuSCs after 48 hours in culture. 6.5K cells were recorded per group. * signifies difference from 12 hours. † signifies difference from 24 hour time point. (p
