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SUPPLEMENTARY INFORMATION
Supplementary Figures
Figure S1 Malic enzyme is required for malate’s protection against DHEA-induced
apoptosis.
S2 cells pretreated with either GFP or malic enzyme (Men) dsRNA were treated with 100
µM DHEA ± 5 mM malate in glucose-free medium for 24 h. Lysates from treated cells
were then analyzed by caspase assay for effector-like caspase activity.
Figure S2 Efficacy of Dronc and Dark dsRNA.
Protein levels of Dronc and Dark after RNAi. Lysates from S2 cells treated with Dronc
dsRNA were analyzed for endogenous Dronc using anti-Dronc antibodies. The efficacy
of Dark RNAi was assessed as previously described (Zimmermann et al, 2002) (Dark1-411,
Dark N-terminal truncate mutant containing CARD domain; asterisk, non-specific band).
Figure S3 Malate does not interfere with DIAP1 degradation.
S2 cells treated with 40 nM DIAP1 dsRNA ± 5 mM malate in serum free Schneider’s
medium were collected at indicated time points for immunoblotting using anti-DIAP1
antibodies (Asterisk, non-specific bands as loading control).
Figure S4 Men RNAi abrogates malate’s protection.
S2 cells pretreated with GFP dsRNA or Men dsRNA pair #2 were treated with 40 μM
CHX ± 5 mM malate in standard Schneider’s medium for 12 h.
Figure S5 The loss of GFP reflects caspase-mediated decrease in cell viability.
Stably transfected S2 cells (see METHODS) were treated with CHX to induce apoptosis.
Cells collected at different time points were used to assess the percentage of GFP-positive
cells by FACS and then lysed to measure cellular DEVDase activity by caspase assay.
Figure S6 Phosphorylation at Dronc S130 does not modulate the interaction between
DIAP1 and Dronc.
S2 cells were transfected to express FLAG-tagged DIAP1 and HA-tagged catalyticallyinactive Dronc with indicated mutations. Co-immunoprecipitation and expression levels
of proteins were performed and examined as described in Fig. 5A. Precipitates on FLAG
beads were analyzed with HA antibodies for DIAP1-bound Dronc.
Figure S7 PKA and CK1 are unlikely involved in malate’s protection against CHXinduced apoptosis.
(A) Neither PKA nor CK1 inhibitors abrogates malate’s protection. S2 cells pretreated
with PKA (30 M H-89) (Zhao et al, 2007) or CK1 (8 M IC261) (Mennella et al, 2009)
inhibitors were treated with 40 µM CHX ± 5 mM malate in standard Schneider’s medium.
Cell density and viability were recorded by pictures. (B) Malate’s protection is not
affected by PKA or CK1 dsRNA. S2 cells were pretreated with GFP, PKA or CK1
dsRNA as described in METHODS. Pretreated cells were then treated with 40 µM CHX
± 5 mM malate to examine malate’s protective effect against CHX-induced cell death.
Figure S8 Overexpression of WT Dronc or Dronc S130A phosphomutant results in
similar eye defects.
Two lines strongly expressing either WT Dronc (WTS) or Dronc S130A phosphomutant
(S130AS) were crossed with DIAP1-deficient flies (Df(3L)st-f13) or flies constitutively
expressing rpr in the eye tissues (GMR-rpr) at 18°C to avoid unnecessary death. Crosses
using flies carrying only Dronc transgenes (1× and 2× Dronc) were incubated at 23°C
instead to enhance eye phenotype. Expression of transgenes as indicated was driven by
GMR-Gal4. Genotype: Driver only (+; GMR-Gal4/+; TM3/+; +), 1× Dronc (+; GMRGal4/+; UAS-Dronc/TM6B; +), 1× Dronc + DIAP1 (+; GMR-Gal4/+; UASDronc/Df(3L)st-f13; +), 1× Dronc + GMR-rpr (+; GMR-Gal4/+; UAS-Dronc/GMR-rpr;
+), 2× Dronc (+; GMR-Gal4/+; UAS-Dronc/UAS-Dronc; +).
Supplementary Table
Table S1. Sequence of oligonucleotide primers used to generate dsRNA templates.
Gene
FlyBase
Annotation
Forward primer (5’ to 3’)
Reverse primers (5’ to 3’)
Dronc
Dark
drICE
DIAP1
Men
CG8091
CG6829
CG7788
CG12284
CG10120
CaMKI
CaMKII
CG1495
CG18069
PKA
CK1
CG4379
CG2028
CTCGAGATTGGAATGCCGAAGAGGCAT
CGCCCAGCGAAAAATGTATTGATTGATG
ATGGACGCCACTAACAATGGAGAATCC
ATGGCATCTGTTGTAGCTGATCTTC
#1F: GGCTGTTACACGAAGAGAAAC
#2F: TACAGTTTGATCCACCAGAAC
TACCCTCCGTTTTATGACGAA
#1F: ATGCTAACCGTTAATCCAAATAAAC
#2F: ATGGCTGCACCAGCAGCCTGTACG
ATGATCGTCCAGCACAAGCCC
ATGGACAAGATGCGGATATTGAAGG
CTGCCCGTGTCGAGATCGCGGTGGGTAAC
CCGAATGCTCTCAGCAATAATGCTTAGTCT
AACCCGTCCGGCTGGAGCCAACTGCTTGT
TCGAAGGTGCGCAGGCG
#1R: ATTGTCACCAGTGCCCCAAAG
#2R: GGCCACCGACGCGGTTCCTTG
CTGCGATGCATTTGTGCTGCC
#1R: TTTTTGGGGTATAAAATCGAATGTAGT
#2R: GGGCTCCTTTTTCAATACCTCAGG
TTCGTAGACGAGTACGCCCAACGC
GCGGAACTTCTTGGCCAGACC
Table S2. Drosophila stocks from the Bloomington Drosophila Stock center (BDRC)
and the Vienna Drosophila RNAi Center (VDRC).
Synonyms
Source / ID
UAS-MEN RNAi hairpins
UAS-G6PDH RNAi hairpins
GMR-rpr
Df(3L)st-f13
VDRC #104016
VDRC #101507
BDRC #5773
BDRC #2993
Supplementary References
Mennella V, Tan DY, Buster DW, Asenjo AB, Rath U, Ma A, Sosa HJ, Sharp DJ (2009)
Motor domain phosphorylation and regulation of the Drosophila kinesin 13, KLP10A. J
Cell Biol 186: 481-490
Zhao Y, Tong C, Jiang J (2007) Hedgehog regulates smoothened activity by inducing a
conformational switch. Nature 450: 252-258
Zimmermann KC, Ricci JE, Droin NM, Green DR (2002) The role of ARK in stressinduced apoptosis in Drosophila cells. J Cell Biol 156: 1077-1087