MCB Accepted Manuscript Posted Online 1 June 2015 Mol. Cell. Biol. doi:10.1128/MCB.00285-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Sestrin2 protects dopaminergic cells against rotenone toxicity through AMPK-dependent
2
autophagy activation
3
Yi-Sheng Hou1, Jun-Jie Guan1, Hai-Dong Xu, Feng Wu, Rui Sheng, Zheng-Hong Qin*
4
Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Jiangsu Key Laboratory of
5
Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Science,
6
Soochow University, Suzhou, China
7 8
Running title: Sestrin2 protects dopaminergic cells through regulating autophagy
9
1
: These authors contribute equally to this work.
10
*: Corresponding author:
11
Zheng-Hong Qin, PhD
12
Department of Pharmacology and Laboratory of Aging and Nervous Diseases, College of Pharmaceutical
13
Science, Soochow University, 199 Ren Ai Road, Suzhou 215123, China
14
Phone: 86-512-65882071
15
Fax: 86-512-65882071
16
Email: [email protected]
17 18 19 20 21 22 23 24 25 26 27 28 29 30 0
31
Abstract
32
Dysfunction of autophagy-lysosomal pathway (ALP) and ubiquitin-proteasome system (UPS) was thought
33
to be an important pathogenic mechanism in synuclein pathology and the Parkinson disease (PD). In the
34
present study, we investigated the role of sestrin2 in autophagic degradation of α-synculein and preservation
35
of cell viability in a rotenone-induced cellular model of PD. We speculated that AMP-activated protein
36
kinase (AMPK) was involved in regulation of autophagy and protection of dopaminergic cells against
37
rotenone toxicity by sestrin2. The results showed that both the mRNA and protein levels of sestrin2 were
38
increased in a TP53-dependent manner in Mes23.5 cells after treatment with rotenone. Genetic knockdown
39
of sestrin2 compromised the autophagy induction in response to rotenone, while over-expression of sestrin2
40
increased the basal autophagy activity. Sestrin2 presumably enhanced autophagy in an AMPK-dependent
41
fashion, as sestrin2 over-expression activated AMPK, and genetic knockdown of AMPK abrogated
42
autophagy induction by rotenone. Restoration of AMPK activity by metformin after sestrin2 knockdown
43
recovered the autophagy activity. Sestrin2 over-expression ameliorated α-synuclein accumulation and
44
inhibited caspase 3 activation and reduced the cytotoxicity of rotenone. These results suggest that sestrin2
45
upregulation attempts to maintain autophagy activity and suppress rotenone cytotoxicity through activation
46
of AMPK, suggesting that sestrin2 exerts a protective effect on dopaminergic cells.
47 48
Key words: Parkinson’s disease; rotenone ; sestrin2; α-synuclein; AMPK
49 50
1
51
Introduction
52
Parkinson disease (PD) is one of the most common neurodegenerative diseases in the aging population. The
53
main pathological feature of PD is the degeneration of midbrain dopaminergic neurons located in the
54
substantia nigra with the presence of ubiquitinated cytoplasmic inclusions named Lewy bodies (LB) in the
55
affected brain regions (1, 2). In vivo and in vitro genetic models over-expressing α-synuclein replicate the
56
essential pathological features of PD (3, 4). α-Synuclein is an aggregate-prone protein, which in pathologic
57
status forms dimeric and higher-order toxic oligomeric species, which then serve as nuclei for the formation
58
of protein aggregates, leading to dysfunction and degeneration of dopaminergic neurons (2, 5, 6).
59
Autophagy, a conserved degradation pathway for proteins, lipids and cellular organelles, is dysregulated
60
in PD (7). Autophagy is both induced and impaired in several genetic and chemical models of PD, leading to
61
an accumulation of immature autophagic vesicles and α-synuclein (8, 9). Enhancement of autophagy with
62
over-expression of beclin1 effectively reduced α-synuclein accumulation and ameliorated PD pathology in
63
animal models (10). Meanwhile, deficiency in autophagy related genes such as atg7 caused PD-like
64
neurodegeneration (11). Thus, autophagy might be protective in response to increased burden of misfolded
65
protein and/or chemical intoxication (8, 12-15). In vivo study with CCI-779, a derivative of rapamycin,
66
retarded the progression of α-synuclein induced neurodegeneration by activation of autophagy, suggesting
67
that mTOR pathway may be a promising target for PD treatment (16). However, pathways regulating
68
autophagy activity under the pathological conditions in PD were not fully elucidated. mTOR (mammalian
69
target of rapamycin) pathway is one of the most characterized pathways responsible for regulation of
70
autophagy, and plays an important role during the pathological development of neurodegenerative diseases
71
including PD (17-21). mTOR senses nutrient and amino acid signals to modulate autophagy activity. AMPK
72
was originally thought to promote autophagy through its ability to inhibit TOR complex 1 (22-24). Recently,
73
evidence proved that association of AMPK with ULK1 (unc-51-like kinase 1) and phosphorylation of ULK1
74
by AMPK was required for autophagy initiation. Meanwhile, TOR complex 1 counteracts the initiation of
75
autophagy through phosphorylation of ULK1 at different sites (25). These results further suggested the
76
functional importance of the co-ordinance of AMPK and TOR signaling in autophagy regulation. It has been
77
reported that pharmacological inhibition of mTOR activated autophagy and produced protective effects in
78
animal models of PD and other neurodegenerative diseases (17, 26).
79
Sestrin2, a stress-response protein, exhibits oxidoreductase activity in vitro and protects cells from
80
oxidative stress. Recent reports showed that sestrin2 could upregulate autophagic catabolism by inhibiting
81
mTOR ( 27, 28, 29). However, how does sestrin2 activate autophagy and its role in PD have not been fully 2
82
understood. Rotenone is an organic pesticide and piscicide (30), which is utilized for production of chemical
83
model of PD. It is highly lipid soluble and freely crosses cellular membranes where it causes mitochondrial
84
dysfunction by inhibiting complex I of the electron transport chain. Subcutaneous rotenone exposure causes
85
highly selective dopaminergic degeneration and α-synuclein aggregation (31). In this study we aimed to
86
investigate if sestrin2 promoted autophagy activity through AMPK and protected dopaminergic cells from
87
rotenone-induced cytotoxicity. The results demonstrated that sestrin2 was upregulated and its induction
88
increased autophagy activity and reduced rotenone cytotoxicity through an AMPK-dependent mechanism.
89
Materials and Methods
90
Antibodies
91
LC3 (1:1,000; Cell signaling #4108, Boston, MA, USA), p62 (1:1,000; American Research Product,
92
12-1107, Waltham, MA, USA), AMPKα (1:1000, Cell signaling #2603), phosphorylated AMPK (Thr172)
93
(1:1000, Cell signaling #2535), caspase3 (1:1000, Cell signaling #9661), phosphor-S6K (1:1000, Abcam
94
ab2571, Cambridge, MA, USA), α-synuclein(1:800, Abcam, ab1903), sestrin2 (1:800, ProteinTech
95
21346-1-AP, Chicago, IL, USA), Flag (1:5000, Sigma F1804, Saint Louis, MO, USA), TP53 (1:1000,
96
Abcam ab183547), Bax (1:1000, Abcam ab5714), actin (1:4000, Sigma A5441).
97
Cell culture and treatment
98
The hybrid Mes 23.5 cell line, derived from somatic cell fusion of rat embryonic mesencephalon cells
99
with murine N18TG2 neuroblastoma cells, were provided by Professor Le, WD from Shanghai Jiaotong
100
University (Shanghai, China) . Mes 23.5 cells display many properties of developing neurons of the SN zona
101
compacta and offer several advantages for such initial studies, including greater homogeneity than primary
102
cultures. Mes 23.5 cells were seeded on polylysine-precoated 24-well plates (Corning, NY, USA) at a
103
density of 104 cells/cm2 and maintained in Dulbecco's modified Eagle's medium (DMEM, GIBCO, NY,
104
USA) containing Sato's components (Sigma) and 5% fetal born serum (Sigma) at 37°C in a 95% air/5% CO2
105
humidified atmosphere incubator.
106
Differentiated PC12 cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of
107
Sciences (Shanghai, China) and were grown in tissue culture flasks at 37°C under an atmosphere of 5% CO2
108
in Dulbecco’s Modified Eagles’s Medium (DMEM) containing 10% fetal bovine serum (complete media).
109
The culture medium was replenished at 2-3 days intervals based on the doubling time. Cells were treated
110
with indicated concentrations of rotenone (Sigma-Aldrich) as previously reported to generate in vitro PD
111
model. For inhibition or activation of autophagy and AMPK, 3-MA, bafilomycin A1, Compound C and
112
metformin were purchased from Sigma-Aldrich. 3
113
Assessment of cell viability
114
Cytotoxicity was measured using MTT assay. Cells were cultured in 96-well plates (1000 cells per well)
115
for 24 h, and then cells were treated with rotenone at indicated concentrations or vehicle for 12-48 h, and
116
then incubated with MTT for 1-2 additional h. The percentage of growth inhibition was calculated as (OD
117
vehicle – OD treated)/OD vehicle x 100%, where OD was measured at 570 nm with a microplate reader
118
(BieTek, Burleigh QLD, Australian). In each experiment, quintuplicate wells were performed for each drug
119
concentration, and assays were repeated in three independent experiments.
120
Quantitative Real-time PCR
121
Total RNA was extracted with the RNAiso Reagent kit (Takara, Dalian, China). cDNA was generated by
122
reverse transcription of 2 μg of total RNA using random primers and PriMescript RT Reagent Kit (Takara) in
123
a total reaction volume of 20 μl according to the manufacturer’s instructions. The sequences of forward and
124
reverse oligonucleotide primers, specific to sestrin2, p62 and housekeep gene (β-actin) were designed using
125
Primer5 software. The primers used are: forward, 5’-CTGGTGACCCCCTCAGCGGATA-3’; reverse,
126
5’-ATGGGCACGGAAGGTTGGGG-3’ for sestrin2, forward, 5’-TGTGGAACATGGAGGGAAGAG-3’;
127
reverse,
128
5’-CTGGCCGAACTCATCCAAG-3’; reverse, 5’- CTGCCTCATGCGTTCCATC-3’ for β-actin. Real-time
129
quantitative PCR was performed in an iCycler 5 (Bio-Rad, Hercules, CA, USA). A 20-fold dilution of each
130
cDNA was amplified in a 20-μl volume, using the Fast Start DNA Master PLUS SYBR Green 1 master mix
131
(Roche Applied Science, Indianapolis, IN, USA), with 200 nM final concentrations of each primer. PCR
132
cycle conditions were 95°C for 10 s, and 35 cycles of 95°C for 15 s and 60°C for 31 s. The amplification
133
specificity was evaluated with melting curve analysis. Threshold cycle Ct, which correlates inversely with
134
the target mRNA levels, was calculated using the second derivative maximum algorithm, the software
135
provided by the iCycler. For each cDNA, the mRNA levels were normalized to β-actin mRNA.
136
RNA interference of TP53, sestrin2 and AMPK
137
Small
5’-TGTGCCTGTGCTGGAACTTTC-3’
interfering
RNAs
(siRNA) Sestrin2,
targeting
for
the
p62
following
GACCATGGCTACTCGCTGA;
and
mRNA:
TP53, α1/α2,
138
GAAGAAAATTTCCGCAAAA
139
AAGAGAAGCAGAAGCACGACGTT. Negative siRNA TAAGGCTATGAAGAGATAC, were synthesized
140
by GenePharma (Shanghai, China). The siRNA oligos used to target AMPK α1/α2 gene were previously
141
validated (32). For transfection, cells were plated in 9-cm dishes at 30% confluence, and siRNA duplexes
142
(200 nM) were introduced into the cells using Lipofectamine 2000 (Invitrogen 11668-019, Carlsbad, CA,
143
USA) according to the manufacturer’s recommendations. 4
AMPK
forward,
144
Expression of GFP-LC3 and sestrin2-Flag
145
The activation of autophagy was detected following transfection of cells with GFP-LC3 and
146
sestrin2-3xFlag expression plasmids. The presence of several intense fluorescent dots in cells is indicative of
147
the accumulation of autophagosomes. Transfection of cells with expression plasmids was performed using
148
Lipofectamine 2000. For each condition, the number of GFP-LC3 dots per cell was determined with a
149
fluorescence microscopy from at least 100 GFP- LC3-positive cells.
150
Sestrin2-3xFlag expression plasmid was generated by PCR from the clone for sestin2 (GC091F05,
151
perchased from Genechem, China) with following primers: cggaattccatgatcgtggcggactccgag (forward) and
152
cg ggatccggtcatgtagcgggtgatggc (reverse), and subsequently digested with EcoR I and BamH I and cloned
153
into the EcoR I and BamH I sites of 3xFlag (Invitrogen). Transfection of cells with expression plasmids was
154
performed using Lipofectamine 2000.
155
Western blot analysis
156
Protein was extracted from cells using cell lysis solution supplemented with protease inhibitors (Roche,
157
04693159001) and phosphorylase inhibitors (Roche, 04906845001). Protein concentration was determined
158
with a BCA protein assay kit (Pierce 23227, Rockford, IL, USA). Equal amounts of proteins were
159
fractionated on Tris-glycine SDS-polyacrylamide gels and subjected to electrophoresis and transferred to NC
160
membranes. Membranes were blocked with TBS containing 5% (w/v) dry milk with 0.1% Tween 20,
161
washed with TBS containing 0.1% Tween 20 (TBST), and then incubated overnight at 4 C with specific
162
primary antibodies in non-fat milk containing 0.1% NaN3. After washing in TBST, membranes were
163
incubated with fluorescence-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch, PA,
164
USA; anti-rabbit, 711-035-152, anti-mouse, 715-035-150) at room temperature for 1 h. Immunoreactivity
165
was detected using ODYSSEY INFRARED IMAGER (Li-COR Biosciences, Lincoln, Nebraska, USA). The
166
signal intensity of primary antibody binding was quantitatively analyzed with Image J software (W.S.
167
Rasband, Image J, NIH, Bethesda, MD) and was normalized to a loading control β-actin.
168
Immunocytochemistry and fluorenscence imaging
169
For immunofluorescence microscopic examination, Mes23.5 cells were plated on 12-mm
170
poly-L-lysine-coated cover slips and cultured for 24 h, then cells were treated with siRNA and drugs. Cells
171
were washed in PBS, fixed with 4% paraformaldehyde in PBS at 4oC for 10 min, and then washed again
172
with PBS. The cells were permeabilized with 0.25% Triton X-100, and were then blocked with 10% normal
173
goat serum (NGS) for 15 min. Primary antibodies: a mouse monoclonal antibody against Flag (Sigma,
174
F1804) diluted in PBS was added to the cells and left for overnight at 4°C. The cover slips were washed 5
175
three times before incubation with secondary antibodies using the same procedure as for the primary
176
antibodies. The cover slips were mounted on slides with mounting medium (Sigma-Aldrich, F4680) and
177
were examined with a laser scanning confocal microscopy (Zeiss, Jena, German).
178
Statistical analysis
179
All data were presented as means ± SEM. Data were subjected to one-way ANOVA using the GraphPad
180
Prism software statistical package (GraphPad Software, San Diego, CA, USA). When a significant group
181
effect was found, post hoc comparisons were performed using the Newman–Keuls t-test to examine special
182
group differences. Independent group t-tests were used for comparing two groups. Significant differences at
183
p
1
Sestrin2 protects dopaminergic cells against rotenone toxicity through AMPK-dependent
2
autophagy activation
3
Yi-Sheng Hou1, Jun-Jie Guan1, Hai-Dong Xu, Feng Wu, Rui Sheng, Zheng-Hong Qin*
4
Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Jiangsu Key Laboratory of
5
Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Science,
6
Soochow University, Suzhou, China
7 8
Running title: Sestrin2 protects dopaminergic cells through regulating autophagy
9
1
: These authors contribute equally to this work.
10
*: Corresponding author:
11
Zheng-Hong Qin, PhD
12
Department of Pharmacology and Laboratory of Aging and Nervous Diseases, College of Pharmaceutical
13
Science, Soochow University, 199 Ren Ai Road, Suzhou 215123, China
14
Phone: 86-512-65882071
15
Fax: 86-512-65882071
16
Email: [email protected]
17 18 19 20 21 22 23 24 25 26 27 28 29 30 0
31
Abstract
32
Dysfunction of autophagy-lysosomal pathway (ALP) and ubiquitin-proteasome system (UPS) was thought
33
to be an important pathogenic mechanism in synuclein pathology and the Parkinson disease (PD). In the
34
present study, we investigated the role of sestrin2 in autophagic degradation of α-synculein and preservation
35
of cell viability in a rotenone-induced cellular model of PD. We speculated that AMP-activated protein
36
kinase (AMPK) was involved in regulation of autophagy and protection of dopaminergic cells against
37
rotenone toxicity by sestrin2. The results showed that both the mRNA and protein levels of sestrin2 were
38
increased in a TP53-dependent manner in Mes23.5 cells after treatment with rotenone. Genetic knockdown
39
of sestrin2 compromised the autophagy induction in response to rotenone, while over-expression of sestrin2
40
increased the basal autophagy activity. Sestrin2 presumably enhanced autophagy in an AMPK-dependent
41
fashion, as sestrin2 over-expression activated AMPK, and genetic knockdown of AMPK abrogated
42
autophagy induction by rotenone. Restoration of AMPK activity by metformin after sestrin2 knockdown
43
recovered the autophagy activity. Sestrin2 over-expression ameliorated α-synuclein accumulation and
44
inhibited caspase 3 activation and reduced the cytotoxicity of rotenone. These results suggest that sestrin2
45
upregulation attempts to maintain autophagy activity and suppress rotenone cytotoxicity through activation
46
of AMPK, suggesting that sestrin2 exerts a protective effect on dopaminergic cells.
47 48
Key words: Parkinson’s disease; rotenone ; sestrin2; α-synuclein; AMPK
49 50
1
51
Introduction
52
Parkinson disease (PD) is one of the most common neurodegenerative diseases in the aging population. The
53
main pathological feature of PD is the degeneration of midbrain dopaminergic neurons located in the
54
substantia nigra with the presence of ubiquitinated cytoplasmic inclusions named Lewy bodies (LB) in the
55
affected brain regions (1, 2). In vivo and in vitro genetic models over-expressing α-synuclein replicate the
56
essential pathological features of PD (3, 4). α-Synuclein is an aggregate-prone protein, which in pathologic
57
status forms dimeric and higher-order toxic oligomeric species, which then serve as nuclei for the formation
58
of protein aggregates, leading to dysfunction and degeneration of dopaminergic neurons (2, 5, 6).
59
Autophagy, a conserved degradation pathway for proteins, lipids and cellular organelles, is dysregulated
60
in PD (7). Autophagy is both induced and impaired in several genetic and chemical models of PD, leading to
61
an accumulation of immature autophagic vesicles and α-synuclein (8, 9). Enhancement of autophagy with
62
over-expression of beclin1 effectively reduced α-synuclein accumulation and ameliorated PD pathology in
63
animal models (10). Meanwhile, deficiency in autophagy related genes such as atg7 caused PD-like
64
neurodegeneration (11). Thus, autophagy might be protective in response to increased burden of misfolded
65
protein and/or chemical intoxication (8, 12-15). In vivo study with CCI-779, a derivative of rapamycin,
66
retarded the progression of α-synuclein induced neurodegeneration by activation of autophagy, suggesting
67
that mTOR pathway may be a promising target for PD treatment (16). However, pathways regulating
68
autophagy activity under the pathological conditions in PD were not fully elucidated. mTOR (mammalian
69
target of rapamycin) pathway is one of the most characterized pathways responsible for regulation of
70
autophagy, and plays an important role during the pathological development of neurodegenerative diseases
71
including PD (17-21). mTOR senses nutrient and amino acid signals to modulate autophagy activity. AMPK
72
was originally thought to promote autophagy through its ability to inhibit TOR complex 1 (22-24). Recently,
73
evidence proved that association of AMPK with ULK1 (unc-51-like kinase 1) and phosphorylation of ULK1
74
by AMPK was required for autophagy initiation. Meanwhile, TOR complex 1 counteracts the initiation of
75
autophagy through phosphorylation of ULK1 at different sites (25). These results further suggested the
76
functional importance of the co-ordinance of AMPK and TOR signaling in autophagy regulation. It has been
77
reported that pharmacological inhibition of mTOR activated autophagy and produced protective effects in
78
animal models of PD and other neurodegenerative diseases (17, 26).
79
Sestrin2, a stress-response protein, exhibits oxidoreductase activity in vitro and protects cells from
80
oxidative stress. Recent reports showed that sestrin2 could upregulate autophagic catabolism by inhibiting
81
mTOR ( 27, 28, 29). However, how does sestrin2 activate autophagy and its role in PD have not been fully 2
82
understood. Rotenone is an organic pesticide and piscicide (30), which is utilized for production of chemical
83
model of PD. It is highly lipid soluble and freely crosses cellular membranes where it causes mitochondrial
84
dysfunction by inhibiting complex I of the electron transport chain. Subcutaneous rotenone exposure causes
85
highly selective dopaminergic degeneration and α-synuclein aggregation (31). In this study we aimed to
86
investigate if sestrin2 promoted autophagy activity through AMPK and protected dopaminergic cells from
87
rotenone-induced cytotoxicity. The results demonstrated that sestrin2 was upregulated and its induction
88
increased autophagy activity and reduced rotenone cytotoxicity through an AMPK-dependent mechanism.
89
Materials and Methods
90
Antibodies
91
LC3 (1:1,000; Cell signaling #4108, Boston, MA, USA), p62 (1:1,000; American Research Product,
92
12-1107, Waltham, MA, USA), AMPKα (1:1000, Cell signaling #2603), phosphorylated AMPK (Thr172)
93
(1:1000, Cell signaling #2535), caspase3 (1:1000, Cell signaling #9661), phosphor-S6K (1:1000, Abcam
94
ab2571, Cambridge, MA, USA), α-synuclein(1:800, Abcam, ab1903), sestrin2 (1:800, ProteinTech
95
21346-1-AP, Chicago, IL, USA), Flag (1:5000, Sigma F1804, Saint Louis, MO, USA), TP53 (1:1000,
96
Abcam ab183547), Bax (1:1000, Abcam ab5714), actin (1:4000, Sigma A5441).
97
Cell culture and treatment
98
The hybrid Mes 23.5 cell line, derived from somatic cell fusion of rat embryonic mesencephalon cells
99
with murine N18TG2 neuroblastoma cells, were provided by Professor Le, WD from Shanghai Jiaotong
100
University (Shanghai, China) . Mes 23.5 cells display many properties of developing neurons of the SN zona
101
compacta and offer several advantages for such initial studies, including greater homogeneity than primary
102
cultures. Mes 23.5 cells were seeded on polylysine-precoated 24-well plates (Corning, NY, USA) at a
103
density of 104 cells/cm2 and maintained in Dulbecco's modified Eagle's medium (DMEM, GIBCO, NY,
104
USA) containing Sato's components (Sigma) and 5% fetal born serum (Sigma) at 37°C in a 95% air/5% CO2
105
humidified atmosphere incubator.
106
Differentiated PC12 cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of
107
Sciences (Shanghai, China) and were grown in tissue culture flasks at 37°C under an atmosphere of 5% CO2
108
in Dulbecco’s Modified Eagles’s Medium (DMEM) containing 10% fetal bovine serum (complete media).
109
The culture medium was replenished at 2-3 days intervals based on the doubling time. Cells were treated
110
with indicated concentrations of rotenone (Sigma-Aldrich) as previously reported to generate in vitro PD
111
model. For inhibition or activation of autophagy and AMPK, 3-MA, bafilomycin A1, Compound C and
112
metformin were purchased from Sigma-Aldrich. 3
113
Assessment of cell viability
114
Cytotoxicity was measured using MTT assay. Cells were cultured in 96-well plates (1000 cells per well)
115
for 24 h, and then cells were treated with rotenone at indicated concentrations or vehicle for 12-48 h, and
116
then incubated with MTT for 1-2 additional h. The percentage of growth inhibition was calculated as (OD
117
vehicle – OD treated)/OD vehicle x 100%, where OD was measured at 570 nm with a microplate reader
118
(BieTek, Burleigh QLD, Australian). In each experiment, quintuplicate wells were performed for each drug
119
concentration, and assays were repeated in three independent experiments.
120
Quantitative Real-time PCR
121
Total RNA was extracted with the RNAiso Reagent kit (Takara, Dalian, China). cDNA was generated by
122
reverse transcription of 2 μg of total RNA using random primers and PriMescript RT Reagent Kit (Takara) in
123
a total reaction volume of 20 μl according to the manufacturer’s instructions. The sequences of forward and
124
reverse oligonucleotide primers, specific to sestrin2, p62 and housekeep gene (β-actin) were designed using
125
Primer5 software. The primers used are: forward, 5’-CTGGTGACCCCCTCAGCGGATA-3’; reverse,
126
5’-ATGGGCACGGAAGGTTGGGG-3’ for sestrin2, forward, 5’-TGTGGAACATGGAGGGAAGAG-3’;
127
reverse,
128
5’-CTGGCCGAACTCATCCAAG-3’; reverse, 5’- CTGCCTCATGCGTTCCATC-3’ for β-actin. Real-time
129
quantitative PCR was performed in an iCycler 5 (Bio-Rad, Hercules, CA, USA). A 20-fold dilution of each
130
cDNA was amplified in a 20-μl volume, using the Fast Start DNA Master PLUS SYBR Green 1 master mix
131
(Roche Applied Science, Indianapolis, IN, USA), with 200 nM final concentrations of each primer. PCR
132
cycle conditions were 95°C for 10 s, and 35 cycles of 95°C for 15 s and 60°C for 31 s. The amplification
133
specificity was evaluated with melting curve analysis. Threshold cycle Ct, which correlates inversely with
134
the target mRNA levels, was calculated using the second derivative maximum algorithm, the software
135
provided by the iCycler. For each cDNA, the mRNA levels were normalized to β-actin mRNA.
136
RNA interference of TP53, sestrin2 and AMPK
137
Small
5’-TGTGCCTGTGCTGGAACTTTC-3’
interfering
RNAs
(siRNA) Sestrin2,
targeting
for
the
p62
following
GACCATGGCTACTCGCTGA;
and
mRNA:
TP53, α1/α2,
138
GAAGAAAATTTCCGCAAAA
139
AAGAGAAGCAGAAGCACGACGTT. Negative siRNA TAAGGCTATGAAGAGATAC, were synthesized
140
by GenePharma (Shanghai, China). The siRNA oligos used to target AMPK α1/α2 gene were previously
141
validated (32). For transfection, cells were plated in 9-cm dishes at 30% confluence, and siRNA duplexes
142
(200 nM) were introduced into the cells using Lipofectamine 2000 (Invitrogen 11668-019, Carlsbad, CA,
143
USA) according to the manufacturer’s recommendations. 4
AMPK
forward,
144
Expression of GFP-LC3 and sestrin2-Flag
145
The activation of autophagy was detected following transfection of cells with GFP-LC3 and
146
sestrin2-3xFlag expression plasmids. The presence of several intense fluorescent dots in cells is indicative of
147
the accumulation of autophagosomes. Transfection of cells with expression plasmids was performed using
148
Lipofectamine 2000. For each condition, the number of GFP-LC3 dots per cell was determined with a
149
fluorescence microscopy from at least 100 GFP- LC3-positive cells.
150
Sestrin2-3xFlag expression plasmid was generated by PCR from the clone for sestin2 (GC091F05,
151
perchased from Genechem, China) with following primers: cggaattccatgatcgtggcggactccgag (forward) and
152
cg ggatccggtcatgtagcgggtgatggc (reverse), and subsequently digested with EcoR I and BamH I and cloned
153
into the EcoR I and BamH I sites of 3xFlag (Invitrogen). Transfection of cells with expression plasmids was
154
performed using Lipofectamine 2000.
155
Western blot analysis
156
Protein was extracted from cells using cell lysis solution supplemented with protease inhibitors (Roche,
157
04693159001) and phosphorylase inhibitors (Roche, 04906845001). Protein concentration was determined
158
with a BCA protein assay kit (Pierce 23227, Rockford, IL, USA). Equal amounts of proteins were
159
fractionated on Tris-glycine SDS-polyacrylamide gels and subjected to electrophoresis and transferred to NC
160
membranes. Membranes were blocked with TBS containing 5% (w/v) dry milk with 0.1% Tween 20,
161
washed with TBS containing 0.1% Tween 20 (TBST), and then incubated overnight at 4 C with specific
162
primary antibodies in non-fat milk containing 0.1% NaN3. After washing in TBST, membranes were
163
incubated with fluorescence-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch, PA,
164
USA; anti-rabbit, 711-035-152, anti-mouse, 715-035-150) at room temperature for 1 h. Immunoreactivity
165
was detected using ODYSSEY INFRARED IMAGER (Li-COR Biosciences, Lincoln, Nebraska, USA). The
166
signal intensity of primary antibody binding was quantitatively analyzed with Image J software (W.S.
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Rasband, Image J, NIH, Bethesda, MD) and was normalized to a loading control β-actin.
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Immunocytochemistry and fluorenscence imaging
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For immunofluorescence microscopic examination, Mes23.5 cells were plated on 12-mm
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poly-L-lysine-coated cover slips and cultured for 24 h, then cells were treated with siRNA and drugs. Cells
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were washed in PBS, fixed with 4% paraformaldehyde in PBS at 4oC for 10 min, and then washed again
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with PBS. The cells were permeabilized with 0.25% Triton X-100, and were then blocked with 10% normal
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goat serum (NGS) for 15 min. Primary antibodies: a mouse monoclonal antibody against Flag (Sigma,
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F1804) diluted in PBS was added to the cells and left for overnight at 4°C. The cover slips were washed 5
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three times before incubation with secondary antibodies using the same procedure as for the primary
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antibodies. The cover slips were mounted on slides with mounting medium (Sigma-Aldrich, F4680) and
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were examined with a laser scanning confocal microscopy (Zeiss, Jena, German).
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Statistical analysis
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All data were presented as means ± SEM. Data were subjected to one-way ANOVA using the GraphPad
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Prism software statistical package (GraphPad Software, San Diego, CA, USA). When a significant group
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effect was found, post hoc comparisons were performed using the Newman–Keuls t-test to examine special
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group differences. Independent group t-tests were used for comparing two groups. Significant differences at
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p
