Transcript Supplementary Material (ppt 10099K)

Figure S1
B
A
B
Figure S1. Infection with fluorescently tagged S. marcescens establish an infection
in the fly’s intestine. To show that oral infection by feeding was efficient, we used the S.
marcescens Db11-GFP strain (gift from D. Ferrandon, IBMC, Strasbourg, France) to show
ingestion and localization of the bacteria in the intestinal lumen. (A) A dissected midgut after
24 hours of oral infection with Db11-GFP. (B) Enlargement of the anterior region. The white
lined area corresponds to the luminal space of the intestinal canal. The white narrows mark
the ingested fluorescent bacteria.
Dorsal-GFP
Relish-RFP
dfoxO-GFP
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
OD600=50
OD600=25
OD600=10
OD600=5
OD600=1
OD600=0.5
5% sucrose
Figure S2
Figure S2
Figure S2. To evaluate the concentration-dependency of Serratia-induced transcription
factor translocation, standardized experiments employing different bacterial concentration
were used for infection.
Transcription factor reporter lines were used and different bacterial concentrations were used. From
top to bottom the different infection conditions are represented, ranging from OD600=0.5 [D, E, F] up to
OD600=50 [S, T, U], all compared to control conditions by ingestion of 5% sucrose only [A, B, C]. The left
column [A, D, G, J, M, P, S] shows Dorsal (NP1-Gal4>pUAST-EGFP-dorsal). In the middle column the
Imd-dependent transcription factor Relish (NP1-Gal4>pUAST-EYFP-relish) is shown [B, E, H, K, N, Q, T].
Here, in E the copper cell region is shown. There was no translocation in the enterocytes at all, but we
first recognized the translocation to the copper cells by an infection with OD600=1. Right figures [C, F, I,
L, O, R, U] show translocation of TF dfoxO (NP1-Gal4>pUAST-foxO-GFP). Translocation of FoxO starts at
low concentrations (OD = 1). Scale bar is 20 µm.
Fly strains
pUAST-EYFP-relish (Tony Ip, Massachusetts)
pUAST-EGFP-dorsal (Tony Ip, Massachusetts)
pUAST foxo GFP (selfmade, Tina)
NP1-GAL4 (D. Ferrandon, Straßburg)
Figure S3
DAPI
A
A‘
B
B‘
C
α-Relish
α-dFoxO
A‘‘
A‘‘‘
B‘‘
B‘‘‘
C‘
C‘‘
C‘‘‘
D
D‘
D‘
‘
D‘‘‘
E
E‘
E‘‘
E‘‘‘
0h
DAPI
2h
4h
6h
8h
G
F
8h
F‘
F‘‘
F‘‘‘
Figure S3
Figure S3. Time-course of the Infection-dependent translocation of dFoxO and Relish in
enterocytes (ECs).
To show that the translocation of FoxO in response to an infection (and the lack of translocation of
Relish) is no artifact of the indicator lines used, we performed time-course experiments employing
immunohistochemistry. In these time course experiment flies from the wild type strain w1118 were
infected with pathogenic bacteria (S. marcescens Db11) for 2 h [B], 4 h [C], 6 h [D], and 8 h [E]. Controls
were only fed with 5% sucrose [A]. The images are representative for enterocytes of the anterior
midgut. Nuclei were stained with Hoechst (blue) [A’-G’]. Antibody-staining was like followed: α-Relish
(red) [A’’-G’]’ and α-dFoxO (green) [A’’’-G’’’]. The co-localization of all staining is represented in [A-G].
Relish is not detectable in the nuclei of the enterocyte at any time point of the experiment. dFoxO
instead shows nuclear translocation after a 8 h infection period [E’’’]. At higher magnification, this
becomes more obvious [G’’’]. Scalebar is 20 µm.
Figure S4
light foxo OE (C45)
60
50
control
control 16°C
foxo-OE
const. 25°C
*
*
40
30
10
*
8
6
*
*
4
*
*
2
lip
4
in
er
ic
or
pt
d3
up
tD
at
tB
at
th
di
pg
rp
-s
c2
0
Figure S4. Relative expression levels of FoxO target genes.
To compare the gene expression after oral infection in wildtype w1118 and foxo-impaired foxo21/21
depicted in figure 1, we here investigated the inducible expression of the same target genes by
constitutive overexpression of foxo in enterocytes (NP1-Gal4 X UAS-foxo, grey bars; control, black bars).
n = 5 for both conditions, statistical analysis was performed with the Mann-Whitney test. Asteriks label
statistically significant differences to matching controls (p<0.05).
Figure S5
Figure S5
Figure S5. Time-course of the Infection-dependent translocation of dFoxO and Relish in
copper cells (CCs).
As shown in figure S3, we employed immunohistochemitry to see the effects of an oral infection
on translocation behavior in copper cells. Flies of the w1118 strain were infected with S.
marcescens Db11 and the translocation of FoxO and Relish was studied. The location of the
transcription factors Relish and dFoxO was detected using specific antibodies. Time course
experiments were performed for 2 h [B], 4 h [C], 6 h [D], 8 h [E] and 24 h [F]. Controls were fed
with 5% sucrose only [A]. Nuclei were stained with Hoechst (blue) [A’-F’]. Antibody-staining was
as following: α-Relish (red) [A’’-F’]’ and α-dFoxO (green) [A’’’-F’’’]. The translocation of Relish in
the Copper cells starts after 6 hours of bacteria feeding [D’’] and is even present after 24 hours
of infection [F’’] in the nucleus. The transcription factor dFoxO is not triggered at any time point
and is remaining in the cytoplasm. Scalebar is 20 µm.
Figure S6
Figure S6. Confirmation of the inducible overexpression using the ternary TARGETexpression system.
To evidence the functionality of induced overexpression using the TARGET expression system
we have done qPCR analysis. For the IMD-signalling pathway we induced the increased
expression of the soluble pattern recognition receptor PGRP-LE and for FoxO-signalling the
transcription factor dFoxO itself. To prove that we have a clear amplification of the target
genes we used gene specific primers for the cDNA synthesis (see above). (A) shows the
efficient expression of PGRP-LE after 24 hours of heatshock activation (29°C) in comparison to
their controls kept at 17°C. We see a hundredfold increased expression of PGRP-LE. Likewise
we see an increased expression of the inducible dFoxO after 24 hours of heatshock activation
in comparison to its control. Only difference is the x-fold change which is only fivefold to
control expression.
Table S2. Influence of dFoxO and IMD on Db11 infection survival.
strain
Db11
(OD600>50)
Median
life span
(d)
w1118
-
42
♂
w1118
+
24.5
♂
dFoxO-KO
-
35
♂
dFoxO-KO
+
11
♂
IMD-KO
-
25
♂
IMD-KO
+
16
w1118
-
44
♀
w1118
+
19
♀
dFoxO-KO
-
45
♀
dFoxO-KO
+
12
♀
IMD-KO
-
25
♀
IMD-KO
+
2.5
sex
♂
♀
Decrease in
Median life span
(% of control)
ChiSquare
(χ2)
p-value
41.76
11.03
0.0009
68.57
28.46
<0.0001
36.00
53.46
<0.0001
56.82
51.38
<0.0001
73.33
54.20
<0.0001
90.00
89.01
<0.0001
Data derived from log rank analysis of the survival curves shown in Fig. 4. Log rank test was
performed using GraphPad Prism 6.00. Experiments shown are representatives of four independent
experiments., each with 10 flies per vial.
Supplemental materials
Infection by feeding
For the concentration-dependent infection experiments (Figure S3), Serratia
marcescens culture we serially diluted to the initial concentration of OD600=50 with
a sterile sucrose solution (5 %) and to the final concentrations with the following
ODs: OD600=50, OD600=25, OD600=10, OD600=5, OD600=5, OD600=1, OD600=0.5.
The bacterial/sucrose solution contained 1 % E133 brilliant blue food coloring dye.
One absorbent filter (37mm; Millipore) was placed into a fly culture tubes and
soaked with 350 µl of the S. marcescens sucrose solution. The flies were then
transferred to these infectious vials for 24 hours at 29°C in a bench incubator with
12/12 h day-night-rhythm.
The intestines were dissected in PBS buffer and then mounted in Roti®-Mount
FluorCare DAPI (Carl Roth GmbH & Co. KG, Karlsruhe, Germany). Images were
captured with the Imager.Z1 equipped with an Apotome (Zeiss, Göttingen,
Germany).
Immunohistochemistry
Immunohistochemistry for Relish and FoxO was performed using standard
protocols and anti-Relish (21F3, DSHB, Iowa, USA) as well as anti-FoxO (Cosmo
Bio, Cologne, Germany) antibodies. DyLight 549 Goat anti mouse and DyLight 488
donkey anti rabbit antibodies (Jackson Immuno Research, Hamburg, Germany)
were used as secondary antibodies.
Oligonucleotide sequences
rpL32 sense CCGCTTCAAGGGACAGTATC – antisense GACAATCTCCTTGCGCTTCT
pgrp-Sc2 sense AACTACCTGAGCTACGCCGTG. – antisense
AGCAGAGGTGAGGGTGTTGGT
thor sense CTCCTGGAGGCACCAAACTT . antisense GAGTTCCCCTCAGCAAGCAA
upd3 sense GAGAACACCTGCAATCTGAA – antisense AGAGTCTTGGTGCTCACTGT
attB sense ACAATCTGGATGCCAAGGTC – antisense TGTCCGTTGATGTGGGAGTA
attD sense AGTGGGGGTCACTAGGGTTC antisense AGGTGATGATTGGCACTTCC
lip4 sense TTCTGATGCAGGGATTGGCA antisense GGGGTAACTTACGCTCACCG
attacin A sense CAATCTGGATGCCAAGGTCT - antisense
TCCCGTGAGATCCAAGGTAG
defensin sense GCTATCGCTTTTGCTCTGCT - antisense
GCCGCCTTTGAACCCCTTGG
diptericin sense GCAATCGCTTCTACTTTGGC - antisense
TAGGTGCTTCCCACTTTCCA
drosomycin sense ACCAAGCTCCGTGAGAACCTT -antisense
TTGTATCTTCCGGACAGGCAG
cecropin C sense AAGATCTTCGTTTTCGTCGC - antisense GTTGCGCAATTCCCAGTC
metchnikowin sense CCACCGAGCTAAGATGCAA - antisense
AATAAATTGGACCCGGTCTTG
drosocin sense GTTCACCATCGTTTTCCTGC - antisense
GGCAGCTTGAGTCAGGTGAT
PGRP-LE sense ACGAGCCACTGCCCTTGCAAC - antisense
AGTCTTACGTTGATCGCCCGC
foxo sense GCATGCACAATGCAAGAGAT - antisense GCCTCGTTATTGAGCACCTC