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Figure legends
Figure 1
A: Wild-type strain (JY333) carrying the pTN54-Atg8 plasmid was cultured in EMM to
OD600nm = 0.5, and then the cells were washed and transferred into sulfur-free EMM.
Thereafter, the cells were harvested at each time point, and Western blot analysis was
performed. The amount of α-tubulin was used as loading control. B: JY333 (WT) and
Δatg1 strains harboring pTN54-Atg8 were cultured in EMM to OD600nm = 0.5, and then
the cells were washed and transferred into EMM (control) and sulfur-free EMM (-S),
10
respectively. Cells were harvested at each time point, and Western blot analysis was
11
performed. C: Wild-type strain (JY333) carrying the pTN54-Atg8 plasmid was cultured
12
in EMM to OD600nm = 0.5, and then the cells were washed and transferred into
13
sulfur-free EMM. After 24 h, cells were subjected to fluorescence microscopy
14
observation. FM4-64 was employed for vacuole staining.
15
16
Figure 2
17
A: JY333 (WT) and Δecl1.2.3 (Δecls) strains were cultured in EMM to OD600nm = 0.5,
18
and then the cells were washed and transferred into EMM (control), sulfur-free (-S), and
19
nitrogen-free (-N) EMM, respectively. Cells were harvested at each time point, and then
20
Western blot analysis was performed. B: JY333 (WT) and Δecl1.2.3 (Δecls) strains
21
were cultured in EMM to OD600nm = 0.5, and then the cells were washed and transferred
22
into EMM and sulfur-free EMM, respectively. Cells were harvested at each time point,
23
and then real-time PCR analysis was performed. The expression of cdc2+ was used as
24
the quantitative control. The results are represented as the mean of three independent
25
experiments with standard deviation.
26
27
Figure 3
28
JY333 (WT) and Δatg1 strains were cultured in SD medium to OD600nm = 1.5, and then
29
the cells were washed and transferred into SD and sulfur-free SD medium, respectively.
30
A: Growth of each strain was monitored at each time point. B: Viability of each strain
31
was measured at each time point. The results are represented as the mean of three
32
independent experiments with standard deviation.
33
10
− S (hour)
12
24
36
48
GFP-Atg8
GFP
α-Tubulin
WT
(hour)
Δatg1
Control
−S
12
12
Control
12
−S
12
GFP-Atg8
GFP
GFP-Atg8
FM4-64
merge
−S
10 μm
Fig.1
10 μm
10 μm
WT
Control
(hour)
12
Δecls
−N
−S
Control
−N
−S
12
12
12
12
12
GFP-Atg8
GFP
−S, 3 hour
−S, 3hour 3
WT
WT
atg4+
−S, 3 hour
−S, 3hour 2
WT
WT
atg13+
−S, 3 hour
−S, 3hour 2
Fig.2
WT
WT
Δecls
Δecls
atg3+
0hour
0 hour
Time0 Control, 3 hour
Control, 3hour −S, 3 hour
−S, 3hour 4
WT
WT
atg8+
Δecls
Δecls
0hour
0hour
0 hour
Time0 Control, 3 hour
Control, 3hour −S, 3 hour
−S, 3hour 4
Δecls
Δecls
0 hour
0hour
Time0 Control, 3 hour
Control, 3hour 5
Δecls
Δecls
0 hour
0hour
Time0 Control, 3 hour
Control, 3hour 3
Relative amount of mRNA
Control, 3 hour
Control, 3hour 4
Relative amount of mRNA
0 hour
Time0 Relative amount of mRNA
atg1+
Relative amount of mRNA
Relative amount of mRNA
Relative amount of mRNA
WT
WT
atg20+
Δecls
Δecls
0hour
0 hour
Time0 Control, 3 hour
Control, 3hour −S, 3 hour
−S, 3hour 3
WT
WT
Δecls
Δecls
OD600nm
10
WT, Control WT, −S Δatg1, Control Δatg1,
Δatg1, −S Δatg1,
50
100
150
200
250
Time (hour)
300
350
400
450
108
100000000
107
10000000
Viability
106
1000000
105
100000
10
10000
WT, Control WT, −S Δatg1, Control Δatg1,
Δatg1, −S Δatg1,
10
1000
10100
Fig.3
50
100
150
200
250
Time (hour)
300
350
400
450
...
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