Supporting figures Figure loss of dfoxO suppressed app-induced small scutellum phenotype

Дата канвертавання24.04.2016
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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 APPCT (e, sd-Gal4/+; UAS-APPCT /+), 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. APPCT 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-APPCT transgenes, APPCT-2 (b, b’, e, e’, h, h’, k and k’) and APPCT-3 (c, c’, f, f’, i, i’, l and l’). m-p are statistical analysis of acridine 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) 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. 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/+).

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