Figures and data in The antimicrobial peptide defensin cooperates with tumour necrosis factor to drive tumour cell death in Drosophila

Version of Record: July 30, 2019

Download
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
- Article PDF
- Figures PDF
Open citations (links to open the citations from this article in various online reference manager services)
- Mendeley
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Jean-Philippe Parvy

Yachuan Yu

Anna Dostalova

Shu Kondo

Alina Kurjan

Philippe Bulet

Bruno Lemaître

Marcos Vidal

Julia B Cordero

(2019)
The antimicrobial peptide defensin cooperates with tumour necrosis factor to drive tumour cell death in Drosophila

eLife 8:e45061.

https://doi.org/10.7554/eLife.45061
- Download BibTeX
- Download .RIS
Cite
Share
CommentOpen annotations (there are currently 0 annotations on this page).

Figures
Tables
Additional files

9 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement

Download asset Open asset

*def* is induced in *dlg* mutant tumour bearing animals.

(A) Working model showing the cooperation between haemocyte-derived TNF and the immune response in the fat body in tumour cell death (Parisi et al., 2014). (B) RT-qPCR analyses showing expression of …
https://doi.org/10.7554/eLife.45061.003

Figure 1—figure supplement 1

Download asset Open asset

*def* mediates animal survival to infection by Gram-positive bacteria.

(A) Survival of *def^sk3* mutant flies (n = 53), upon Gram-positive, *Listeria innocua* infection, is compared to wild-type (wt) (n = 55) and *spatzle (spz^M7)* (n = 30) (the Toll ligand) mutant flies. (B) …
https://doi.org/10.7554/eLife.45061.004

Figure 2 with 1 supplement

Download asset Open asset

Def restricts tumour growth and promotes tumour cell death.

(**A-E’**) Quantification of tumour volume (TV) (A) and tumour cell death (TCD) (B) in wing imaginal discs from *dlg^40.2* (n = 17), *dlg^40.2;def^sk3* (n = 19) and *dlg^40.2;def^sk4* (n = 20) mutant larvae and …
https://doi.org/10.7554/eLife.45061.005

Figure 2—figure supplement 1

Download asset Open asset

Tumour suppression by Def is independent of infection and tumour cell proliferation.

(**A-E’**) Quantification of TV (A) and tumour proliferation (B) from *dlg^40.2* (n = 19), *dlg^40.2;def^sk3* (n = 16) and *dlg^40.2;def^sk4* (n = 17) mutant larvae and representative pictures of the corresponding …
https://doi.org/10.7554/eLife.45061.006

Figure 3

Download asset Open asset

Def from the trachea and the fat body mediates tumour cell death.

(A) RT-qPCR analysis of *def* expression in gut, fat body and trachea dissected from *w¹¹¹⁸* or *dlg^40.2* mutant larvae (n = 3). **B-C**, Fat body (B) and trachea (C) from *dlg^40.2* mutant larvae stained with …
https://doi.org/10.7554/eLife.45061.007

Figure 4 with 1 supplement

Download asset Open asset

Def produced by immune tissues specifically targets tumour cells.

(**A-A’’**) DAPI (blue), anti-Dcp1 (green) and anti-HA antibody (red) staining of *dlg^40.2* mutant tumour from larvae overexpressing a *def-HA* construct in the fat body and the trachea (*dlg^40.2;lpp >UAS-def…*
https://doi.org/10.7554/eLife.45061.008

Figure 4—figure supplement 1

Download asset Open asset

Characterisation of UAS-def-HA expression.

(**A-B**) DAPI (blue) and anti-HA antibody (red) staining showing basal expression of Def-HA in trachea (A) but not in fat body (B) of *UAS-def-HA* larvae. **C-D**, DAPI (blue) and anti-HA antibody (red) …
https://doi.org/10.7554/eLife.45061.009

Figure 5

Download asset Open asset

Imd pathway activation is required for *def* expression and Def-mediated tumour cell death.

(A) RT-qPCR analysis of *def* expression from *dlg^40.2* and *dlg^40.2;imd¹* mutant larvae (n = 4). **B-E’**, Quantification of TV (B) and TCD (C) in wing imaginal discs from *dlg^40.2* (n = 15) and *dlg^40.2;imd¹* …
https://doi.org/10.7554/eLife.45061.010

Figure 6

Download asset Open asset

Imd and Toll pathway activation are required in the trachea and the fat body to promote Defensin-dependent tumour cell death.

(A) RT-qPCR analysis of *def* expression from *dlg^40.2* mutant larvae (*dlg^40.2;btl>*) expressing a *ctrl-IR* or *imd-IR* in the trachea (n = 3). **B-E’**, Quantification of TV (B) and TCD (C) from *dlg^40.2* mutant …
https://doi.org/10.7554/eLife.45061.011

Figure 7 with 3 supplements

Download asset Open asset

TNF is required for PS exposure and Defensin-driven tumour cell death.

(**A-A’’**) DAPI (blue), Annexin V (green) and anti-Def (red) staining of *dlg^40.2* mutant tumours. (**B-B’’**) Enlargement of inset from **A’’** (white outline) showing Annexin V (B), Def (B’) and merged …
https://doi.org/10.7554/eLife.45061.012

Figure 7—figure supplement 1

Download asset Open asset

Def-HA does not associate to PS-negative *egr*-mutant tumours.

(**A-B**) DAPI (blue) and anti-HA antibody (red) staining showing expression of Def-HA in trachea (A) and fat body (B) of *dlg^40.2;egr³;lpp-gal4 >UAS* def-HA larvae. **C-C’**, DAPI (blue), anti-Dcp1 (green) …
https://doi.org/10.7554/eLife.45061.013

Figure 7—figure supplement 2

Download asset Open asset

TNF signalling activation does not require functional *def*.

(**A-C’**) DAPI (white), anti-Dcp1 (red) and anti-Matrix metalloproteinase 1 (Mmp1, a reporter of JNK pathway activation, green) antibody staining of wing disc from *dlg^40.2*, *dlg^40.2;def^sk3* and *dlg^40.2;de…*
https://doi.org/10.7554/eLife.45061.014

Figure 7—figure supplement 3

Download asset Open asset

*scrib* mutant tumours expose PS in a TNF-dependent manner and are sensitive to Def action.

(**A-A’**) DAPI (blue), anti-Dcp1 (red) and Annexin V (green) staining of *scrib¹* mutant tumours. (**B-B’**) DAPI (blue), anti-Dcp1 (red) and Annexin V (green) staining of egr³;*scrib¹* double mutant tumours. …
https://doi.org/10.7554/eLife.45061.015

Author response image 1

Download asset Open asset

Anti-tumoural immunity in flies: the key players.

(**A, B**) Third instar larval imaginal wing disc overexpressing of *hep^wt* using *engrailed-gal4, UAS-rfp (eng>rfp; hep^wt*) (A; red). Discs where stained with AnnexinV to visualize PS exposure (B; green). …

Author response image 2

Download asset Open asset

Anti-tumoural immunity in flies: the key players.

(A, B) Third instar larval imaginal wing disc overexpressing of *hep^act* using *rutond-gal4 (rn>hep^act*).

Discs where stained with anti-Dcp1 to visualize apoptotic cell death (B; red).

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (Drosophila melanogaster)	w¹¹¹⁸	(Dewey et al., 2004)	BDSC: 3605; RRID:BDSC_3605
Genetic reagent (Drosophila melanogaster)	w¹¹¹⁸ iso	(Ferreira et al., 2014)	N/A
Genetic reagent (Drosophila melanogaster)	dlg^40.2/FM7	(Mendoza-Topaz et al., 2008)	Flybase_FBal0240608
Genetic reagent (Drosophila melanogaster)	FRT82B,scrib¹/TM6	(Bilder et al., 2000)	Flybase_FBal0103577
Genetic reagent (Drosophila melanogaster)	egr³	(Igaki et al., 2002)	Flybase_FBal0147163
Genetic reagent (Drosophila melanogaster)	imd¹	(Leulier et al., 2000)	Flybase_ FBal0045906
Genetic reagent (Drosophila melanogaster)	rel^E20	(Leulier et al., 2000)	DGGR: 109927; RRID:DGGR_109927
Genetic reagent (Drosophila melanogaster)	def^sk3	(Hanson et al., 2019)	N/A
Genetic reagent (Drosophila melanogaster)	def^sk4	(Hanson et al., 2019)	N/A
Genetic reagent (Drosophila melanogaster)	btl-gal4,UAS-RFP/CyO	Irene Miguel-Aliaga	N/A
Genetic reagent (Drosophila melanogaster)	lpp-gal4/TM6B	(Brankatschk and Eaton, 2010)	N/A
Genetic reagent (Drosophila melanogaster)	tub-gal4	Bloomington Drosophila Stock Center	BDSC: 5138; RRID:BDSC_5138	y(1) w[*]; P{w[+mC]=tubP-GAL4}LL7/TM3, Sb(4) Ser(1)
Genetic reagent (Drosophila melanogaster)	hml^Δ-gal4,UAS-gfp	Bruno Lemaitre	N/A
Genetic reagent (Drosophila melanogaster)	en-gal4	Bloomington Drosophila Stock Center	BDSC: 30564; RRID:BDSC_30564	y1 w; P{w + mW.hs=en2.4 GAL4}e16E*
Genetic reagent (Drosophila melanogaster)	UAS-def IR	Vienna Drosophila Resource Centre	VDRC: 102437; RRID:Flybase_FBst0474306	P{KK111656}VIE-260B
Genetic reagent (Drosophila melanogaster)	UAS-imd IR	Vienna Drosophila Resource Centre	VDRC: 101834; RRID:Flybase_FBst0473707	P{KK109863}VIE-260B
Genetic reagent (Drosophila melanogaster)	UAS-myd88 IR	Vienna Drosophila Resource Centre	VDRC: 25402; RRID:Flybase_FBst0455868	w¹¹¹⁸; P{GD9716}v25402
Genetic reagent (Drosophila melanogaster)	UAS-dlg IR	Vienna Drosophila Resource Centre	VDRC: 41136; RRID:Flybase_FBst0463952	w¹¹¹⁸; P{GD4689}v41136/TM3
Genetic reagent (Drosophila melanogaster)	UAS-egr IR	Vienna Drosophila Resource Centre	VDRC: 108814; RRID:Flybase_FBst0480608	P{KK103432}VIE-260B
Genetic reagent (Drosophila melanogaster)	UAS-w IR	Bloomington Drosophila Stock Center	BDSC: 25785; RRID:BDSC_25785	y(1) v(1); P{y[+t7.7] v[+t1.8]=TRiP.JF01786}attP2
Genetic reagent (Drosophila melanogaster)	UAS-def	(Tzou et al., 2000)	Flybase_FBal0145092
Genetic reagent (Drosophila melanogaster)	UAS-def-3xHA	FlyORF	FlyORF: F002467; RRID:Flybase_FBal0298643	M{UAS-Def.ORF.3xHA.GW}ZH-86Fb
Genetic reagent (Drosophila melanogaster)	UAS-dcr2	Bloomington Drosophila Stock Center	BDSC: 24650; RRID:BDSC_24650	w[1118]; P{w[+mC]=UAS-Dcr-2.D}2
Genetic reagent (Drosophila melanogaster)	spz^M7	(Neyen et al., 2014)	N/A
Antibody	Anti-GFP (Chicken polyclonal)	Abcam	Cat# ab13970; RRID:AB_300798	IF(1:4000)
Antibody	Anti-Def (Mouse polyclonal)	Dahua Chen (Ji et al., 2014)	N/A	IF(1:100)
Antibody	Anti-HA (Mouse monoclonal)	Cell Signaling Technology	Cat# 2367, RRID:AB_10691311	IF(1:1000)
Antibody	Anti-dcp1 (Rabbit polyclonal)	Cell Signaling Technology	Cat# 9578, RRID:AB_2721060	IF(1:100)
Antibody	Anti-phospho-Histone H3 (Ser10) (Rabbit polyclonal)	Cell Signaling Technology	Cat# 9701, RRID:AB_331535	IF(1:100)
Antibody	Anti-phospho-Histone H3 (Ser28) (Rabbit polyclonal)	Cell Signaling Technology	Cat# 9713, RRID:AB_823532	IF(1:100)
Antibody	Anti-Mmp1 (Mouse clonality unknown)	Developmental Studies Hybridoma Bank	Cat# 3B8D12, RRID:AB_579781	IF(1:10)
Antibody	Anti-Chicken IgY Alexa 488 (Goat polyclonal)	Molecular Probes	Cat# A-11039, RRID:AB_142924	IF(1:500)
Antibody	Anti-Mouse IgG Alexa 488 (Goat polyclonal)	Molecular Probes	Cat# A-11029, RRID:AB_138404	IF(1:500)
Antibody	Anti-Mouse IgG Alexa 594 (Goat polyclonal)	Molecular Probes	Cat# A-11032, RRID:AB_141672	IF(1:500)
Antibody	Anti-Rabbit IgG Alexa 488 (Goat polyclonal)	Molecular Probes	Cat# A-11008, RRID:AB_143165	IF(1:500)
Antibody	Anti-Rabbit IgG Alexa 594 (Goat polyclonal)	Thermo Fischer Scientific	Cat# A-11037, RRID:AB_2534095	IF(1:500)
Sequence-based reagent	rpl32-fwd	This paper	PCR primers	AGGCCCAAGATCGTGAAGAA
Sequence-based reagent	rpl32-rev	This paper	PCR primers	TGTGCACCAGGAACTTCTTGA
Sequence-based reagent	def-fwd	This paper	PCR primers	CTTCGTTCTCGTGGCTATCG
Sequence-based reagent	def-rev	This paper	PCR primers	ATCCTCATGCACCAGGACAT
Sequence-based reagent	def-PCR-fwd	This paper	PCR primers	TTATTGCAGAAACGGGCTCT
Sequence-based reagent	def-PCR-rev	This paper	PCR primers	ATGGTAAGTCGCTAACGCTAATG
Sequence-based reagent	def-seq	This paper	Sequencing primers	CGTGTCTTCCTGCACAGAAA
Sequence-based reagent	attA-fwd	This paper	PCR primers	ATGCTCGTTTGGATCTGACC
Sequence-based reagent	attA-rev	This paper	PCR primers	TCAAAGAGGCACCATGACCAG
Sequence-based reagent	cecA1-fwd	This paper	PCR primers	CTCAGACCTCACTGCAATAT
Sequence-based reagent	cecA1-rev	This paper	PCR primers	CCAACGCGTTCGATTTTCTT
Sequence-based reagent	dro-fwd	This paper	PCR primers	CGTTTTCCTGCTGCTTGCTT
Sequence-based reagent	dro-rev	This paper	PCR primers	GGCAGCTTGAGTCAGGTGAT
Sequence-based reagent	drs-fwd	This paper	PCR primers	CTCTTCGCTGTCCTGATGCT
Sequence-based reagent	drs-rev	This paper	PCR primers	ACAGGTCTCGTTGTCCCAGA
Peptide, recombinant protein	Drosophila endogenous Defensin	Bulet EIRL	N/A
Peptide, recombinant protein	Drosophila Synthetic Defensin	Genepep	N/A	ATCDLLSKWNWNHTACAGHCIAKGFKGGYCNDKAVCVCRN
Commercial assay or kit	High Capacity cDNA Reverse Transcription Kit	Applied Biosystems	Cat# 4368813
Commercial assay or kit	PerfeCTa SYBR Green FastMix (Low ROX)	Quanta Bio	Cat# 95074–012
Commercial assay or kit	TRIzol Reagent	Thermo Fisher Scientific	Cat# 15596018
Commercial assay or kit	Turbo DNA free Kit	Life Technologies LTD	Cat# AM1907
Commercial assay or kit	High Capacity cDNA Reverse Transcription Kit	Applied Biosystems	Cat# 4368813
Chemical compound, drug	4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI)	Sigma	Cat# D9542	1 μg/mL
Software, algorithm	Fiji	NIH	https://fiji.sc/
Software, algorithm	GraphPad Prism 6	GraphPad	RRID:SCR_002798
Software, algorithm	7500 Real-Time PCR Software	Applied Biosystems	RRID:SCR_014596
Software, algorithm	BatchQuantify	(Johansson et al., 2019)	https://github.com/emltwc/2018-Cell-Stem-Cell
Software, algorithm	GraphPad Prism 6	GraphPad	RRID:SCR_002798
Software, algorithm	Volocity 3D Image Analysis Software	Perkin Elmer	RRID:SCR_002668
Software, algorithm	ZEN two lite	Zeiss	RRID:SCR_013672
Other	RNasine Plus RNase Inhibitor	Promega	Cat# N261
Other	Vectashield mounting media	Vector Laboratories, Inc.	Cat# H-1000–10
Other	Annexin V, Alexa Fluor 568 conjugate	Life Technologies LTD	Cat# A13202	1:20
Other	Annexin V, Alexa Fluor 488 conjugate	Life Technologies LTD	Cat# A13201	1:20
Other	Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	Cat# 15140122	Pen: 100 IU/mL Strep: 100 mg/mL
Other	PfuUltra II Fusion HS DNA Polymerase	Agilent	Cat# 600670
Other	AnnexinV binding buffer	Fisher Scientific	Cat# BDB556454