Supporting figures
Figure S1. Loss of dfoxO suppressed APP-induced small scutellum phenotype
Light images of Drosophila adult thorax are shown. Compared with the control (a,
ap-Gal4/UAS-GFP), ectopic expression of APP driven by ap-Gal4 produced a small
scutellum phenotype (b, ap-Gal4 UAS-APP/+), which was suppressed by expressing
two independent dfoxO RNAi (c, ap-Gal4 UAS-APP/+; UAS-dfoxO-IR-1/+; d,
ap-Gal4 UAS-APP/+; UAS-dfoxO-IR-2/+).
Figure S2. Loss of dfoxO suppressed APP-induced small wing phenotype
Light images of adult wing are shown. Expression of APP (b, sd-Gal4/+; UAS-APP/+),
but not APP△CT (e, sd-Gal4/+; UAS-APP△CT /+), driven by sd-Gal4 resulted in a small
wing phenotype, which was suppressed by mutation (c, sd-Gal4/+; UAS-APP/+;
dfoxOΔ94/+) or RNAi down-regulation (d, sd-Gal4/+; UAS-APP/+; UAS-dfoxO-IR-1/+)
of dfoxO.
Figure S3. APP△CT failed to induce cell death in the neuronal and non-neuronal
tissues of Drosophila
Fluorescent images of acridine orange staining (a-c, a’-c’, d-f, g-i, j-l and j’-l’) of the
central nervous system (a-c), ventral nerve cord (a’-c’), eye (d-f), notum (g-i) and
wing disc (j-l, j’-l’) from 3rd instar larvae and light images of adult eye (d’-f’) or
thorax (g’-i’) are shown. elav-Gal4 (a-c, a’-c’), GMR-Gal4 (d-f and d’-f’) or
pnr-Gal4 (g-i and g’-i’) and ptc-Gal4 (j-l and j’-l’) was used to dive the expression of
two additional UAS-APP△CT transgenes, APP△CT-2 (b, b’, e, e’, h, h’, k and k’) and
APP △ CT-3 (c, c’, f, f’, i, i’, l and l’). m-p are statistical analysis of acridine
1
orange-positive cells in a-c, d-f, g-i and j-l, respectively. Error bars means ±SEM，n.s.:
no significant difference.
Figure S4. Quantitative analysis of dfoxO mRNA expression
RT-PCR analysis of dfoxO mRNA expression in 3rd instar larvae was shown. The
larvae were heat shocked in 37℃ for 1 hour and recovered for 2 hours in 25℃.
Figure S5. APP induced dFoxO-dependent cell death in Drosophila
a-d, Light images of adult wings are shown. The areas for anterior cross vein (acv)
are boxed on the left and enlarged on the right. Expression of GFP (a,
ptc-Gal4/UAS-GFP)
or
two
independent
dfoxO
RNAi
(c,
ptc-Gal4/+;
UAS-dfoxO-IR-1/+ and d, ptc-Gal4/+; UAS-dfoxO-IR-2/+), or heterozygous mutation
in dFoxO (b, ptc-Gal4/+; dfoxOΔ94/+) produced no obvious phenotype in the adult
wing. e-j, Acridine orange staining of wing discs from 3rd instar larvae are shown.
Lower panels are high magnification of boxed areas in the upper panels. Compared to
the control (e, ptc-Gal4/UAS-GFP), APP-induced cell death (f, ptc-Gal4 UAS-APP/+)
was suppressed by the expression of dfoxO RNAi (g, ptc-Gal4 UAS-APP /+;
UAS-dfoxO-IR-2/+). Mutation in dfoxO (h, ptc-Gal4/+; dfoxOΔ94/+) or expression of
two independent dfoxO RNAi (i, ptc-Gal4/+; UAS-dfoxO-IR-1/+ and j, ptc-Gal4/+;
UAS-dfoxO-IR-2/+) had no effect on cell death by themselves. k. Statistical analysis
of acridine orange-positive cells in e-j. Error bars means ±SEM，***: P≤0.001.
Figure S6. APP induced hid transcription in Drosophila
a-c, X-Gal staining of a hid-LacZ reporter gene in 3rd instar wing discs. sd-Gal4 (a)
2
was used to drive the expression of GFP (b) or APP/APLP2 (c).
Figure S7. Physical interaction between AICD and FoxO proteins in human cells
AICD co-localized with FoxO1 in HEK 293T cells before (upper panels) or after
(lower panels) H2O2 treatment. Cells were transfected with AICD-Myc and
FoxO1-GFP, stained with Hoechst and incubated with anti-Myc antibodies.
Immunoreactivity was detected with IgG conjugated to Alexa Fluor 546 (red, detects
AICD).
Figure S8. AICD does not affect the cellular localization of FoxO proteins
The nuclear accumulation of FoxO1, FoxO3a and FoxO4A3 in 293T cells in the
presence or absence of AICD was shown. A minimum of 200 cells per condition were
counted. n.s.: no significant difference.
Figure S9. APPswe and APP-C99 promotes FoxO4A3 induced Bim expression
APPswe and C99 cooperated with FoxO4A3 to stimulate luciferase expression driven
by Bim promoter. **: P≤0.01, ***: P≤0.001. HEK 293T cells were transfected with
BIM-luciferase reporter (firefly, 0.3 μg), pRL-null (Renilla, 0.01 μg), and empty
vector or the indicated plasmids. Activity of the firefly luciferase was normalized to
that of the Renilla luciferase.
Figure S10. PS-1 promotes FoxO-induced Bim expression
Expression of PS-1 up-regulated FoxO-dependent Bim-luciferase expression. HEK
293T cells were transfected with Bim-luciferase reporter (firefly, 0.3 μg), pRL-null
(Renilla, 0.01 μg) and the indicated effector plasmids, using empty vector as control.
3
Activity of the firefly luciferase was normalized to that of the Renilla luciferase.
Figure S11. The expression of Bim is up-regulated in frontal lobe of APP/PS1
transgenic mice
Frontal lobes were dissected from 7-month-old wild type or APP/PS1 transgenic mice
(Jackson Laboratory; #004462). Lysates were subjected to immunoblotting with
antibodies against Bim or actin, and the relative expression level of Bim was
normalized to that of actin.
Video S1. Locomotion of a control larva (Appl-Gal4/+; UAS-GFP/+).
Video S2. Expression of APP induced a larva locomotion defect (Appl-Gal4/+;
UAS-APP/+).
Video S3. Loss of dFoxO suppressed APP-induced larva locomotion defect
(Appl-Gal4/+; UAS-APP/+; dFoxOΔ94/+).
4