1
2
3 ESM-Methods
Electronic Supplementary Material
4 Real-time quantitative PCR and western blot analysis
5 We used the Trizol
TM
reagent (Invitrogen, USA) and followed the
6
manufacturer’s instructions to isolate total RNA [1]. The purity and concentration of
7 each RNA sample was estimated using the Nanodrop 2000/2000C (Thermo Scientific).
8 Approximately 1 μg of total RNA from each sample was used to generate first-stand
9 cDNA using an oligo-dT (15) primer (Promega) and M-MLV reverse transcriptase
10 (Promega) according to the manufacturer's protocol.
Fluorescent real-time
11 quantitative PCR was performed in a 10-μL total reaction volume comprising 5 μL of
12 a premix (BIO-RAD, SsoFast TMEvaGreen®Supermix), 0.5 μL of each primer (2 μM)
13 for quail or 1 μL of each primer (2 μM) for other species, 1 μL of cDNA template, and
14 water. PCR was conducted using a Mx3000P detection system (Stratagene) and the
15 parameters were as follows: 30 s at 95°C, followed by 35 cycles of 10 s at 95 °C, 10 s
16 at 60 °C, and 25 s at 72 °C (for
P. sinensis ); or 32 cycles of 10 s at 95 °C, 10 s at
17
59 °C, and 25 s at 72 °C (for
T. septentrionalis ); or 35 cycles of 10 s at 95 °C, 10 s at
18 60 °
º
C, and 5 s at 72 °C (for C. coturnix ); or 35 cycles of 10 s at 95 °C, 10 s at 57 °C,
19 and 20 s at 72 °C (for
A. platyrhynchos ). A melting curve of products was performed
20 after amplification to exclude non-specific products by one cycle of 30 s at 95 °C, 30
21 s at 65 °C, and 30 s at 95 °C. Each reaction was performed in triplicate and
22 normalized using
β
-actin. The specific primers used in the PCR reaction are shown in
23 Table S1.
1

24 Table S1. Primer sequences used in real-time quantitative PCR
25
26
Species Gene
Primer sequence (5ˊ→3ˊ)
β-actin
TGAACCCCAAAGCCAACAGA
GGGCGTAGCCTTCGTAGATG
Takydromus
Septentrionalis
hsp70
GCAAGGAACTCAACAAAAGCA
CTCGATCCCCAGCGACAG accession No.
Designed according to the conserved domain of other species
Designed according to the conserved domain of other species
β-actin
CGCTCGTCGTTGATAATGG
Pelodiscus
TTCAGGGTCAGGATACCTCTTT
sinensis
hsp70
GCTCAACGCCGACCTCTT
CGCCCACCAGCACAATCTC
Coturnix
coturnix
β-actin
hsp70
CCAATGCCATCCTGCGTC
TCTCGGCTGTGGTGGTGAA
CCCACGGCTGCTGCTATT
CGCCTCACTGCTCGCTTA
Anas
platyrhynchos
β-actin
hsp70
AY998617.1
JF694990.1
AF199488.1
EU622852.1
ATCTTTCTTGGGTATGGAGTC
GAAGCATTTGCGGTGG
EF667345.1
GGATGATAAACTGAAGGGAAAG
AAGCAAAGAATGGATTGACTGT
EU678246.2
Livers were homogenized in ice-cold buffer [40 mM Tris (pH 7.5), 1 mM PMSF,
27 1 mM EDTA] to extract total protein. The homogenates were centrifuged at 10,000 g
28 for 15 min at 4 °C and protein concentrations in the supernatants were measured using
29 a Nanodrop 2000/2000C (Thermo Scientific). Western blot analysis was performed
30
according to the procedure reported by Huang and Kang [2], with some modifications.
31 Samples were boiled for 10 min with a loading buffer (5:1) containing sodium
32 dodecyl sulfate (10%), β-mercaptoethanol (5%), bromophenol blue (0.5%), Tris (0.25
33 M, pH 6.8), and glycerin (50%). For each species, equal amounts of proteins were
2

34 loaded onto a 10% SDS-PAGE gel, together with a protein molecular weight ladder
35 (PageRuler Pre-stained protein ladders, Thermo Scientific, 26619). After
36 electrophoresis at 90V for 2 h, one gel was stained with Coomassie Blue and the
37 others transferred onto a nitrocellulose membrane using a Semi-Dry cell (JUNYI).
38 The gel subjected to Coomassie Blue was stained by 0.5% G250 in 10% ethanoic acid
39 and 40% methanol. After staining, the gel was washed with destaining solution (30%
40 methanol and 10% ethanoic acid) and then photographed. The total protein content
41 was quantified as the main band intensity using the image analysis software (Quantity
42 One, Bio-Rad). For the electroblotted one, membrane was blocked with 3% skim milk
43 for 1 h and incubated overnight with primary anti-HSP70 monoclonal antibody (BD;
44 610607 for birds and lizard, antibody prepared in our lab for the turtle samples) at a
45 dilution of 1:2,000. After washing in TBST buffer (10 mM Tris, 0.87% NaCl, 0.05%
46 Tween-20, pH 7.5), horseradish peroxidase-conjugated anti-mouse IgG (Kang Wei,
47 CW0102 for birds and lizards) or horseradish peroxidase-conjugated anti-rabbit IgG
48 (KPL, 14-16-06 for turtles) were added at a dilution of 1:5,000. Then the membrane
49 was washed with TBST again and the bound antibody was detected using a solution
50 consisting of 6 μL of 30% H
2
O
2
, 9 mL TBS, and 6 mg 4-chloro-1 naphthol in
51 methanol. The membrane was scanned and stored. HSP70 protein levels were
52 quantified as the band intensity as described above.
53
54
55
References:
1. Krivoruchko, A., Storey, K.B. 2010 Regulation of the heat shock response
56 under anoxia in the turtle, Trachemys scripta elegans . J Comp Physiol B 180 ,
3

57
58 2.
59
60
61
403-414.
Huang, L.-H., Kang, L. 2007 Cloning and interspecific altered expression of heat shock protein genes in two leafminer species in response to thermal stress.
Ins Mol Biol 16 , 491-500.
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62
63 Fig. S1. Detection of the plasmids and the expression of PsHSP70 in the hatchlings of
64 Pelodiscus sinensis . (A). hsp70 gene expression.
Hatchlings from eggs injected with
65 pIRESPsHsp70 (lane1) or pIRES2-EGFP (lane 2 and 3) were used. (a) Extracted
66 DNA was used for PCR with primers specific for pIRESPsHsp70 (lane 1 and 2) or
67 specific for pIRES2-EGFP (lane 3); (b) Extracted RNA was used for PCR with the
68 primers described in (a); (c) Extracted RNA was used for PCR with primers specific
69 to
β
-actin (internal control). (B). Distribution and expression of plasmids. Hatchlings
70 from eggs injected with either PBS (a, b, c), pIRES2-EGFP (d, e, f) or pIRESPsHsp70
71 (g, h, i) were fixed and observed under fluorescence microscope for DAPI nuclear
72 staining (a, d, g) with an exposure time of 20 ms or green fluorescence with an
73 exposure time of 200 ms (b, e, h). (C). HSP70 expression in hatchlings from eggs
74 injected with PBS, pIRES2-EGFP or pIRESPsHsp70. The loading amounts of total
75 protein were all 400 μg. HSP70 expression was quantified as the quotient between
76 HSP70 band intensities and total protein intensities (the loading control of Coomassie
77 Blue staining, LC) in each lane. Bands of LC were shown above the western blots.
78
Data are shown as means ± SE (n = 3 for each treatment). Means with different letters
79 above the error bars are statistically different (Tukey’s post-hoc test). The level of
80 HSP70 was significantly higher in hatchlings from eggs injected with pIRESPsHsp70
81 than those from eggs injected with pIRES2-EGFP or PBS ( F
2,6
= 227.9, P < 0.0001).
82
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84
85
86
87
6

97
98
99
100
101
102
88
89
90 Fig. S2. The hatching success of Pelodiscus sinensis embryos from different HSP70
91 expression treatments. The eggs from HSP70 overexpression treatments were injected
92 with pIRESPsHsp70, while those from shame control with pIRES2-EGFP. Statistical
93 significance between groups was calculated by X 2 test (***, P < 0.001). Numbers
94 above the error bars are sample size.
95
96
100
80
60
40
20
0
41/79
* * *
74/95
Sham control HSP70 overexpression
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103
104
105
106
107
108
109
110
Fig. S3. The influence of HSP70 overexpression during embryogenesis on body size
(A), swimming performance (B), and specific growth rate (C) of Pelodiscus sinensis hatchlings. The eggs from HSP70 overexpression treatments were injected with 10 μg of plasmid pIRES2-EGFP cloned with turtle hsp70 sequences. The specific growth rate (SGR) was calculated as SGR = (lnW t
− lnW o
)/T × 100%, in which W o
= initial body mass; W t
= final body mass; and T = duration of experiment.
Data are shown as means

SE. Numbers above the error bars are sample size in the bottom graph and are applicable to the upper graphs of this figure.
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