1. Developmental Biology
  2. Immunology and Inflammation

Muscle function and homeostasis require cytokine inhibition of AKT activity in Drosophila

  1. Katrin Kierdorf  Is a corresponding author
  2. Fabian Hersperger
  3. Jessica Sharrock
  4. Crystal M Vincent
  5. Pinar Ustaoglu
  6. Jiawen Dou
  7. Attila Gyoergy
  8. Olaf Groß
  9. Daria E Siekhaus
  10. Marc S Dionne  Is a corresponding author
  1. Imperial College London, United Kingdom
  2. University of Freiburg, Germany
  3. Institute of Science and Technology, Austria
https://doi.org/10.7554/eLife.51595
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Cite this article
  1. Katrin Kierdorf
  2. Fabian Hersperger
  3. Jessica Sharrock
  4. Crystal M Vincent
  5. Pinar Ustaoglu
  6. Jiawen Dou
  7. Attila Gyoergy
  8. Olaf Groß
  9. Daria E Siekhaus
  10. Marc S Dionne
(2020)
Muscle function and homeostasis require cytokine inhibition of AKT activity in Drosophila
eLife 9:e51595.
https://doi.org/10.7554/eLife.51595
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4 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
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Dome inhibition in adult muscle reduces lifespan, disrupts homeostasis, and causes AKT hyperactivation.

(A) STAT activity in different muscles in 10xSTAT92E-GFP reporter fly. One fly out of 5 shown. Upper panel: lateral view, Scale bar = 500 µm. Lower panels: dorsal thorax (left); dorsal abdomen (middle); tibia (right), Scale bar = 100 µm. (B) Lifespan of 24B-Gal80ts/+ and 24B-Gal80ts > dome∆ at 29°C. Log-Rank test: χ2 = 166, ***p<0.0001; Wilcoxon test: χ2 = 157.7, ***p<0.0001. (C) Negative geotaxis assay of 14-day-old 24B-Gal80ts/+ and 24B-Gal80ts > dome∆ flies. Points represent mean height climbed in individual vials (~20 flies/vial), pooled from three independent experiments. Unpaired T-test: **p=0.0033. (D) Muscle (Phalloidin) and neutral lipid (LipidTox) of thorax samples from 14-day-old 24B-Gal80ts/+ and 24B-Gal80ts > dome∆ flies. One representative fly per genotype is shown of six analysed. Scale bar = 50 µm. (E) Thin layer chromatography (TLC) of triglycerides in 7-day-old 24B-Gal80ts/+ and 24B-Gal80ts > dome∆ flies, n = 3–4 per genotype. One experiment of two is shown. Unpaired T-Test: ***p<0.0001. (F) Glucose and trehalose (left) and glycogen (right) in 7-day-old 24B-Gal80ts/+ and 24B-Gal80ts > dome∆ flies, pooled from two independent experiments. Unpaired T-Test (Glucose +Trehalose): ***p<0.0001 and unpaired T-Test (Glycogen): ***p<0.0001. (G) CO2 produced, O2 consumed, and RQ of 7-day-old 24B-Gal80ts/+ and 24B-Gal80ts > dome∆ flies. Box plots show data from one representative experiment of three, with data collected from a 24 hr measurement pooled from 3 to 4 tubes per genotype with 10 flies/tube. P values from Mann-Whitney test. (H–L) Western blots of leg protein from 14-day-old 24B-Gal80ts/+ and 24B-Gal80ts > dome∆ flies. (H) Phospho-AKT (S505). One experiment of four is shown. Unpaired T-Test: ***p<0.0001. (I) Total AKT. One experiment of two is shown. Unpaired T-Test: **p=0.0017. (J) Phospho-p70 S6K (T398). One experiment of two is shown. Unpaired T-Test: ns p=0.0539. (K) Phospho-AMPKα (T173). One experiment of three is shown. Unpaired T-Test: ns p=0.1024. (L) Phospho-ERK (T202/Y204). One experiment of three is shown. Unpaired T-Test: ns p=0.0826.

Figure 1—figure supplement 1
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Further characterisation of the requirement for dome in adult muscle.

(A) STAT activity (10xSTAT92E-GFP) and muscle (MHC-RFP) colocalize in adult flies. One fly of 6 shown. Scale bar = 500 µm. (B) dome expression by qRT-PCR in thorax samples from 14-day-old 24B-Gal80ts/+ and 24B-Gal80ts > dome∆ flies. Unpaired T-Test: *p=0.0102. (C) Lifespan of UAS-dome∆/+ and 24B-Gal80ts > dome∆ at 29°C. Log-Rank test: χ2 = 100.8, ***p<0.0001; Wilcoxon test: χ2 = 76.2, ***p<0.0001. (D) Lifespan of 24B-Gal80ts/+ and 24B-Gal80ts > dome∆ at 25°C. Log-Rank test: χ2 = 61.83, ***p<0.0001; Wilcoxon test: χ2 = 55.18, ***p<0.0001. (E) Smurf assay of 14-day-old 24B-Gal80ts/+ (n = 49) and 24B-Gal80ts-dome∆ flies (n = 18). Data pooled from two independent experiments. (F) Lifespan of Mef2-Gal80ts/+ and Mef2-Gal80ts > dome∆ flies at 29°C. Log-Rank test: χ2 = 86.96, ***p<0.0001; Wilcoxon test: χ2 = 78.61, ***p<0.0001. (G) Negative geotaxis assay of 14-day-old Mef2-Gal80ts/+ and Mef2-Gal80ts > dome∆ flies. Points represent mean climbing height of individual vials analysed (~20 flies/vial), pooled from three independent experiments. Unpaired T-Test: ***p<0.0001. (H, I) Western blots of protein from legs of 14-day-old Mef2-Gal80ts/+ and Mef2-Gal80ts > dome∆ flies. One of three independent experiments is shown. (H) Phospho-AKT. Unpaired T-Test: *p=0.0145. (I) Total AKT. Unpaired T-Test: ***p<0.0003. (J) Western blots of Phospho-AKT in leg samples from 14-day-old MHC-Gal4/+ and MHC-Gal4 >dome∆ (II) flies. One of two independent experiments is shown. Unpaired T-Test: ns p=0.2557. (K) Lifespan of MHC-Gal4/+ and MHC-Gal4 >dome∆ (II) flies at 29°C. Log-Rank test: χ2 = 82.9, ***p<0.0001; Wilcoxon test: χ2 = 58.91, ***p<0.0001. (L–R) Expression by qRT-PCR of Akt1 and insulin-like peptides in whole fly samples from 14-day-old 24B-Gal80ts/+ and 24B-Gal80ts-dome∆ flies. All transcript levels are normalized to Rpl1 and shown in arbitrary units [au]. P values in B, G, H-J, L-R from unpaired T-test. (S) Feeding assay of 24-Gal80ts/+ and 24B2-Gal80ts > dome∆ flies at 29°C. n = 4 were analysed per genotype. Absorbance at 620 nm is shown in arbitrary units [au]. Unpaired T-Test: ns p=0.8819.

Figure 2 with 1 supplement
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Hop is required, but not sufficient, for Dome to control AKT.

(A) Lifespan of w1118 and hopTum-L flies at 29°C. Log-Rank test: χ2 = 0.3223, ns p=0.5702; Wilcoxon test: χ2 = 0.4756, ns p=0.4906. (B) Phospho-AKT in leg samples from 14-day-old w1118 and hopTum-L flies. One experiment of two is shown. Unpaired T-Test: ns p=0.6854. (C) Actin (Phalloidin) and neutral lipid (LipidTox) in flight muscle from 14-day-old w1118 and hopTum-L flies. One representative fly shown of six analysed per genotype. Scale bar = 50 µm. (D) Lifespan of 24B-Gal80ts/+, 24B-Gal80ts > dome∆, and hopTum-L;24B-Gal80ts > dome∆ flies at 29°C. Log-Rank test (24B-Gal80ts/+ vs. 24B-Gal80ts > dome∆): χ2 = 319.4, ***p<0.0001; Wilcoxon test (24B-Gal80ts/+ vs. 24B-Gal80ts > dome∆): χ2 = 280.2, ***p<0.0001. Log-Rank test (24B-Gal80ts/+ vs. hopTum-L 24B-Gal80ts > dome∆): χ2 = 18.87, ***p<0.0001; Wilcoxon test (24B-Gal80ts/+ vs. hopTum-L 24B-Gal80ts > dome∆): χ2 = 20.83, ***p<0.0001. (E) Phospho-AKT in leg samples from 14-day-old 24B-Gal80ts/+, 24B-Gal80ts > dome∆ and hopTum-L;24B-Gal80ts > dome∆ flies. P values from unpaired T-Test.

Figure 2—figure supplement 1
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Interactions of dome with AMPK, MAPK, and FOXO signalling in adult muscle.

(A) Phospho-AKT in leg samples from 14-day-old 24B-Gal80ts/+, 24B-Gal80ts > AMPKα-IR, and 24B-Gal80ts > AMPKβ-IR flies. One of three independent experiments is shown. P values from unpaired T-Test. (B) Phospho-AKT in leg samples from 14-day-old 24B-Gal80ts/+, 24B-Gal80ts > rl IR, and 24B-Gal80ts > Dsor1 IR flies. One of three independent experiments is shown. P values from unpaired T-Test. (C) Lifespan of 24B-Gal80ts/+, 24B-Gal80ts > rl IR, and 24B-Gal80ts > Dsor1 IR flies at 29°C. Log-Rank test (24B-Gal80ts/+ vs. 24B-Gal80ts > rl IR): χ2 = 60.29, ***p<0.0001; Wilcoxon test (24B-Gal80ts/+ vs. 24B-Gal80ts > rl IR): χ2 = 58.32, ***p<0.0001; Log-Rank test (24B-Gal80ts/+ vs. 24B-Gal80ts > Dsor1 IR): χ2 = 1.186, ns p=0.2760; Wilcoxon test (24B-Gal80ts/+ vs. 24B-Gal80ts > Dsor1 IR): χ2 = 0.0033, ns p=0.9538. (D) Phospho-AKT in leg samples from 14-day-old 24B-Gal80ts/+ and 24B-Gal80ts > hop IR flies. One of two independent experiments is shown. Unpaired T-Test: *p=0.0187. (E) Lifespan of 24B-Gal80ts/+ and 24B-Gal80ts > hop IR flies at 29°C. Log-Rank test (24B-Gal80ts/+ vs. 24B-Gal80ts > hop IR): χ2 = 546.4, ***p<0.0001; Wilcoxon test (24B-Gal80ts/+ vs. 24B-Gal80ts > hop IR): χ2 = 458.1, ***p<0.0001. P values in A, C, E from unpaired T-test.

Figure 3 with 1 supplement
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AKT hyperactivation causes pathology in 24B-Gal80ts > dome∆ flies.

(A) Lifespan of 24B-Gal80ts/+ and 24B-Gal80ts > myr-AKT at 29°C. Log-Rank test: χ2 = 115.5, ***p<0.0001; Wilcoxon test: χ2 = 123.6, ***p<0.0001. (B) Negative geotaxis assay of 14-day-old 24B-Gal80ts/+ and 24B-Gal80ts > myr-AKT flies. Points represent mean height climbed in individual vials (~20 flies/vial), pooled from two independent experiments. Unpaired T-Test: *p=0.018. (C) TLC of triglycerides in 7-day-old 24B-Gal80ts/+ and 24B-Gal80ts > myr-AKT flies, n = 3–4 per genotype. One experiment of two is shown. Unpaired T-Test: *p=0.0144. (D) Glucose and trehalose (left panel) and glycogen (right panel) in 7-day-old 24B-Gal80ts/+ (n = 12) and 24B-Gal80ts > myr-AKT (n = 9) flies, pooled from two independent experiments. Unpaired T-Test (Glucose +Trehalose): ***p=0.0009 and unpaired T-Test (Glycogen): ***p<0.0001. (E) CO2 produced, O2 consumed, and RQ of 7-day-old 24B-Gal80ts/+ and 24B-Gal80ts > myr-AKT flies. Box plots show data from one representative experiment of three, with data points collected from a 24 hr measurement pooled from 3 to 4 tubes per genotype with 10 flies/tube. P values from Mann-Whitney test. (F) Phalloidin and LipidTox staining of thorax samples from 14-day-old 24B-Gal80ts/+ and 24B-Gal80ts > myr-AKT flies. One representative fly per genotype is shown of 3 analysed per group in two independent experiments. Scale bar = 50 µm. (G) Lifespan of 24B-Gal80ts/+, 24B-Gal80ts > dome∆, UAS-domeΔ/+, 24B-Gal80ts > AKT-IR, UAS-AKT-IR/+, 24B-Gal80ts > AKT-IR;dome∆ and UAS-AKT-IR;dome∆/+ flies at 29°C. Log-Rank test (24B-Gal80ts > dome∆ vs. 24B-Gal80ts > AKT-IR;dome∆): χ2 = 101.0, ***p<0.0001; Wilcoxon test (24B-Gal80ts > dome∆ vs. 24B-Gal80ts > AKT-IR;dome∆): χ2 = 59.87, ***p<0.0001. (H) Lifespan of 24B-Gal80ts/+, 24B-Gal80ts > dome∆, foxo-GFP;24B-Gal80ts/+, and foxo-GFP;24B-Gal80ts > dome∆ flies at 29°C. Log-Rank test (24B-Gal80ts > dome∆ vs. foxo-GFP;24B-Gal80ts > dome∆): χ2 = 114.0, ***p<0.0001; Wilcoxon test (24B-Gal80ts > dome∆ vs. foxo-GFP;24B-Gal80ts > dome∆): χ2 = 93.59, ***p<0.0001. (I) Glucose + trehalose and glycogen in 7-day-old 24B-Gal80ts/+, 24B-Gal80ts > domeΔ, foxo-GFP;24B-Gal80/+, and foxo-GFP; 24B-Gal80ts > domeΔ flies. Statistical testing was performed with one-way ANOVA. (J) TLC of triglycerides in 7-day-old 24B-Gal80ts/+, 24B-Gal80ts > domeΔ, foxo-GFP;24B-Gal80ts/+, and foxo-GFP;24B-Gal80ts > domeΔ flies. Statistical testing was performed with one-way ANOVA.

Figure 3—figure supplement 1
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Mutual regulation by AKT, Foxo, and Dome.

(A) dome expression by qRT-PCR in whole fly samples from 14-day-old 24B-Gal80ts/+, 24B-Gal80ts > dome∆, 24B-Gal80ts > Akt-IR, and 24B-Gal80ts > Akt-IR;dome∆ flies. (B–F) Gene xpression analysis of dome, upd2, Pepck, Thor and InR in whole fly samples from 14-day-old 24B-Gal80ts/+, 24B-Gal80ts > dome∆, foxo-GFP;24B-Gal80ts/+, and foxo-GFP;24B-Gal80ts > dome∆ flies. P values from unpaired T-test. (B) dome. (C) upd2. (D) Pepck. (E) Thor. (F) InR. (G) Western blot for GFP to detect the Foxo-GFP fusion protein in leg samples from 14-day-old 24B-Gal80ts/+, 24B-Gal80ts > dome∆, foxo-GFP;24B-Gal80ts/+, and foxo-GFP;24B-Gal80ts > dome∆ flies. (H) Western blot for Phospho-AKT in leg samples from 14-day-old 24B-Gal80ts/+, 24B-Gal80ts > dome∆, foxo-GFP;24B-Gal80ts/+, and foxo-GFP;24B-Gal80ts > dome∆ flies. One of two independent experiments is shown. P values in A-D from unpaired T-test. (I–M) Gene expression analysis of Akh and six confirmed or putative Akh target genes in whole fly samples from 14-day-old 24B-Gal80ts/+ and 24B-Gal80ts > dome∆ flies. Transcript levels are shown in arbitrary units [au]. P values from unpaired T-test. (I) Akh. (J) Hsl. (K) bmm. (L) plin1. (M) plin2.

Figure 4 with 2 supplements
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Plasmatocytes promote muscle Dome activity.

(A) Muscle (MHCYFP) and plasmatocytes (srpHemo-3xmCherry) in 7-day-old flies. Plasmatocytes are found in close proximity to adult muscles. One representative fly of 5 is shown. Scale bar = 500 µm. (B) Legs and plasmatocytes in 7-day-old 10xSTAT92E-GFP;srpHemo-3xmCherry flies. Muscle with high JAK-STAT activity (green) is surrounded by plasmatocytes (magenta). One representative fly of 5 is shown. Scale bar = 100 µm. (C) STAT activity and plasmatocytes in legs from control (10xSTAT92E-GFP;crq-Gal4 >CD8-mCherry/+) and upd3-overexpressing (10xSTAT92E-GFP;crq-4>CD8mCherry/UAS-upd3) flies. One representative fly of 10–14 is shown. Scale bar = 100 µm. Graph shows mean fluorescence intensity (MFI). Unpaired T-Test: ***p<0.0001. (D) STAT activity and plasmatocytes in legs from control (10xSTAT92E-GFP;crq-Gal80ts > CD8-mCherry/+) and plasmatocyte-depleted (10xSTAT92E-GFP;crq-Gal80ts > CD8 mCherry/rpr) flies. One representative fly of six is shown. Scale bar = 250 µm. (E) Western blot analysis of STAT-driven GFP in legs from 7-day-old control (10xSTAT92E-GFP;crq-Gal80ts > CD8-mCherry/+) and plasmatocyte-depleted (10xSTAT92E-GFP;crq-Gal80ts > CD8 mCherry/rpr flies). One representative experiment of three is shown. Graph shows STAT-GFP/α-tubulin for control (crq-Gal80ts/+) and plasmatocyte-depleted (crq-Gal80ts > rpr) leg samples. Unpaired T-Test: *p=0.0121. (F) Lifespan of crq-Gal80ts/+, crq-Gal80ts > rpr, upd2Δ upd3Δ;crq-Gal80ts/+, and upd2Δ upd3Δ;crq-Gal80ts > rpr flies at 29°C. Log-Rank test (crq-Gal80ts/+ vs. crq-Gal80ts > rpr): χ2 = 101.7, ***p<0.0001; Wilcoxon test (crq-Gal80ts/+ vs. crq-Gal80ts > rpr): χ2 = 107.8, ***p<0.0001; Log-Rank test (crq-Gal80ts/+ vs. upd2 Δ upd3Δ;crq-Gal80ts > rpr): χ2 = 60.03, ***p<0.0001; Wilcoxon test (crq-Gal80ts/+ vs. upd2 Δ upd3Δ;crq-Gal80ts > rpr): χ2 = 80.97, ***p<0.0001. (G) Actin (Phalloidin) and neutral lipid (LipidTox) in thorax samples from 14-day-old crq-Gal80ts/+, crq-Gal80ts > rpr, upd2 Δ upd3Δ;crq-Gal80ts/+, and upd2 Δ upd3Δ;crq-Gal80ts > rpr flies. One representative fly per genotype shown of 6 analysed per group. Scale bar = 50 µm. (H) Lifespan of crq-Gal4/+ and crq-Gal4 >upd1 IR flies at 29°C. Log-Rank test: χ2 = 31.36, ***p<0.0001; Wilcoxon test: χ2 = 22.17, ***p=0.0001. (I) Expression by qRT-PCR of upd1, upd2 and upd3 in thorax samples of crq-Gal4/+ and crq-Gal4 >upd1 IR flies, data from four independent samples of each genotype.. Unpaired T-Test (upd1): ns p=0.848, unpaired T-Test (upd2): *p=0.0449 and unpaired T-Test (upd3): **p=0.0038. (J) Lifespan of crq-Gal4/+, upd2 Δ upd3Δ;UAS-upd1-IR/+, and upd2Δ upd3Δ;crq-Gal4 >upd1 IR flies at 29°C. Log-Rank test (crq-Gal4/+ vs. upd2 Δ upd3Δ;crq-Gal4 >upd1 IR): χ2 = 41.12, ***p<0.0001; Wilcoxon test (crq-Gal4/+ vs. upd2Δ upd3Δ;crq-Gal4 >upd1 IR): χ2 = 54.47, ***p<0.0001 Log-Rank test (crq-Gal4/+ vs. upd2 Δ upd3Δ;UAS-upd1-IR/+): χ2 = 14.46, ***p<0.0001; Wilcoxon test (crq-Gal4/+ vs. upd2Δ upd3Δ;UAS-upd1-IR/+): χ2 = 19.99, ***p<0.0001. P values in C, E, H from unpaired T-test.

Figure 4—figure supplement 1
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Further characterisation of plasmatocyte-depleted flies.

(A) Plasmatocyte depletion in crq-Gal80ts > rpr flies (right) and controls (left) at 24 hr, 48 hr and 72 hr at 29°C. One out of four flies is shown. Scale bar = 250 µm. (B) Quantification of mCherry-positive plasmatocytes in crq-Gal80ts > rpr flies (right) and controls (left) by flow cytometry after 72 hr at 29°C. n = 3 per group was analysed. Unpaired T-Test: *p=0.0337. (C) Negative geotaxis assay of 14-day-old crq-Gal80ts/+, crq-Gal80ts > rpr, upd2∆upd3∆;UAS-rpr/+ and upd2∆upd3∆;crq-Gal80ts > rpr flies. Points represent mean climbing height of individual vials analysed (~20 flies/vial). Unpaired T-Test: *p=0.0194.

Figure 4—figure supplement 2
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Further characterisation of requirements for specific Upds.

(A) Glucose + trehalose and glycogen in 7-day-old crq-Gal80ts/+, crq-Gal80ts > rpr, upd2Δ upd3Δ;crq-Gal80ts/+, and upd2Δ upd3Δ; crq-Gal80ts > rpr flies. (B) TLC of triglyceride in 7-day-old crq-Gal80ts/+, crq-Gal80ts > rpr, upd2Δ upd3Δ;crq-Gal80ts/+, and upd2Δ upd3Δ;crq-Gal80ts > rpr flies, n = 2–3 samples per genotype. (C) Western blot analysis of STAT-driven GFP in legs from 7-day-old w1118, upd3Δ, upd2Δ, and upd2Δ upd3Δ flies. One representative experiment of two is shown. (D) STAT activity and plasmatocytes in legs from 7-day-old control (crq-Gal4/+), upd1 knockdown (crq-Gal4 >upd1 IR), and upd3 knockdown (crq-Gal4 >upd3 IR) flies. One representative fly is shown of 5–7 imaged for each genotype. Scale bar = 100 µm. Mean fluorescence intensity (MFI) is shown for all flies imaged. (E) Western blot analysis of STAT-driven GFP in legs from 7-day-old control (crq-Gal4/+), upd1 knockdown (crq-Gal4 >upd1 IR), and upd3 knockdown (crq-Gal4 >upd3 IR) flies. One of two independent experiments is shown. (F) Western blot analysis of STAT-driven GFP in thorax from 7-day-old crq-Gal4/+, crq-Gal4 >upd1, upd2Δupd3Δ;crq-Gal4/+ and upd2Δupd3Δ;crq-Gal4 >upd1 IR flies. P values in A-F from unpaired T-test.

Tables

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent
(D. melanogaster)		w1118; tubulin-Gal80ts/SM6a;24B-Gal4/TM6c, Sb1This studyInserted Elements: P[w[+mC]=tubP-GAL80[ts]]; P[GawB]how24B
Genetic reagent (D. melanogaster)w1118; tubulin-Gal80ts/SM6a;Mef2-Gal4/TM6c, Sb1This studyInserted Elements: P[w[+mC]=tubP-GAL80[ts]]; P[GAL4-Mef2.R]3
Genetic reagent (D. melanogaster)w1118;;UAS-dome∆/TM6c, Sb1Brown et al., 2001Gift of James Castelli-Gair Hombría
Genetic reagent (D. melanogaster)w1118;UAS-dome∆/CyOBrown et al., 2001Gift of James Castelli-Gair Hombría
Genetic reagent (D. melanogaster)w1118;;UAS-myr-AKT/TM6c, Sb1Stocker et al., 2002Gift of Ernst Hafen
Genetic reagent (D. melanogaster)w;UAS-AMPKα-IRVienna Drosophila Research Center (VDRC)RRID:FlyBase_FBst0478025; VDRC 106200
Genetic reagent (D. melanogaster)w;UAS-AMPKβ-IRVDRCRRID:FlyBase_FBst0476347; VDRC 104489
Genetic reagent (D. melanogaster)w;UAS-rl-IRVDRCRRID:FlyBase_FBst0480887; VDRC 109108
Genetic reagent (D. melanogaster)w;UAS-Dsor1-IRVDRCRRID:FlyBase_FBst0479098; VDRC 107276
Genetic reagent (D. melanogaster)w1118;foxoGFPBDSCRRID:BDSC_38644Inserted Element:
PBac[foxo-GFP.FLAG]VK00037
Genetic reagent (D. melanogaster)w;UAS-AKT-IRVDRCRRID:FlyBase_FBst0475561; VDRC 103703
Genetic reagent (D. melanogaster)w1118;10xSTAT92E-GFPBDSC
Bach et al., 2007		RRID:BDSC_26197Inserted Element:
P[10XStat92E-GFP]1
Genetic reagent (D. melanogaster)w1118;MHC-Gal4,MHC-RFP/SM6aBDSCRRID:BDSC_38464Inserted Element:
P[Mhc-GAL4.F3-580]2; P[Mhc-RFP.F3-580]2
Genetic reagent (D. melanogaster)w upd2 Δ upd3Δ;;;BDSCRRID:BDSC_55729
Genetic reagent (D. melanogaster)w1118;;crq-Gal4/TM6 c, Sb1Gift of Nathalie Franc
Genetic reagent (D. melanogaster)w1118;tub-Gal80ts;TM2/TM6 c, Sb1BDSCRRID:BDSC_7108
Genetic reagent (D. melanogaster)w1118;;UAS-rpr/TM6 c, Sb1BDSCRRID:BDSC_5824
Genetic reagent (D. melanogaster)w1118;UAS-CD8-mCherryBDSCRRID:BDSC_27391
Genetic reagent (D. melanogaster)w1118;;srpHemo-3xmCherry/TM6c, Sb1Gyoergy et al., 2018
Genetic reagent (D. melanogaster)w;UAS-hop-IRVDRCRRID:FlyBase_FBst0463355; VDRC 40037
Genetic reagent (D. melanogaster)w;UAS-upd1-IR/SM6aVDRCRRID:FlyBase_FBst0459787; VDRC 3282
Genetic reagent (D. melanogaster)w;UAS-upd3-IRVDRCRRID:FlyBase_FBst0456774; VDRC 27134
Genetic reagent (D. melanogaster)w1118;;UAS-upd3/TM6 c, Sb1Gift of Bruce Edgar
Genetic reagent (D. melanogaster)w1118;UAS-2xeGFP/SM6aBDSCRRID:BDSC_6874
Genetic reagent (D. melanogaster)w1118 hopTum-L/FM7hBDSCRRID:BDSC_8492backcrossed onto w1118 background
Sequence-based reagentAkt1_forwardThis studyPCR primers5’-ctttgcgagtattaactggacaga-3’
Sequence-based reagentAkt1_reverseThis studyPCR primers5’-ggatgtcacctgaggcttg-3’
Sequence-based reagentIlp2_forwardThis studyPCR primers5’-atcccgtgattccaccacaag-3’
Sequence-based reagentIlp2_reverseThis studyPCR primers5’-gcggttccgatatcgagtta-3’
Sequence-based reagentIlp3_forwardThis studyPCR primers5’-caacgcaatgaccaagagaa-3’
Sequence-based reagentIlp3_reverseThis studyPCR primers5’-tgagcatctgaaccgaact-3’
Sequence-based reagentIlp4_forwardThis paperPCR primers5’-gagcctgattagactgggactg-3’
Sequence-based reagentIlp4_reverseThis paperPCR primers5’-tggaccggctgcagtaac-3’
Sequence-based reagentIlp5_forwardThis paperPCR primers5’-gccttgatggacatgctga-3’
Sequence-based reagentIlp5_reverseThis paperPCR primers5’-agctatccaaatccgcca-3’
Sequence-based reagentIlp6_forwardThis paperPCR primers5’-cccttggcgatgtatttcc-3’
Sequence-based reagentIlp6_reverseThis paperPCR primers5’-cacaaatcggttacgttctgc-3’
Sequence-based reagentIlp7_forwardThis paperPCR primers5’-cacaccgaggagggtctc-3’
Sequence-based reagentIlp7_reverseThis paperPCR primers5’-caatatagctggcggacca-3’
Sequence-based reagentdome_forwardThis paperPCR primers5’-cggactttcggtactccatc-3’
Sequence-based reagentdome_reverseThis paperPCR primers5’-accttgatgaggccaggat-3’
Sequence-based reagentupd1_forwardThis paperPCR primers5’-gcacactgatttcgatacgg-3’
Sequence-based reagentupd1_reverseThis paperPCR primers5’- ctgccgtggtgctgtttt −3’
Sequence-based reagentupd2_forwardThis paperPCR primers5’-cggaacatcacgatgagcgaat-3’
Sequence-based reagentupd2_reverseThis paperPCR primers5’-tcggcaggaacttgtactcg-3’
Sequence-based reagentupd3_forwardThis paperPCR primers5’-actgggagaacacctgcaat-3’
Sequence-based reagentupd3_reverseThis paperPCR primers5’-gcccgtttggttctgtagat-3’
Sequence-based reagentPepck_forwardThis paperPCR primers5’-ggataaggtggacgtgaag-3’
Sequence-based reagentPepck_reverseThis paperPCR primers5’-acctcctgcgaccagaact-3’
Sequence-based reagentThor_forwardThis paperPCR primers5’-caggaaggttgtcatctcgga-3’
Sequence-based reagentThor_reverseThis paperPCR primers5’-ggagtggtggagtagagggtt-3’
Sequence-based reagentInR_forwardThis paperPCR primers5'-gcaccattataaccggaacc-3'
Sequence-based reagentInR_reverseThis paperPCR primers5'-ttaattcatccatgacgtgagc-3'
Sequence-based reagentAkh_forwardThis paperPCR primers5’- agccgtgctcttcatgct-3’
Sequence-based reagentAkh_reverseThis paperPCR primers5’-aaaggttccaggaccagctc-3’
Sequence-based reagentHsl_forwardThis paperPCR primers5’-cttggaaatacttgaggggttg-3’
Sequence-based reagentHsl_reverseThis paperPCR primers5’-agatttgatgcagttctttgagc-3’
Sequence-based reagentbmm_forwardThis paperPCR primers5’-gtctcctctgcgatttgccat-3’
Sequence-based reagentbmm_reverseThis paperPCR primers5’-ctgaagggacccagggagta-3’
Sequence-based reagentplin1_forwardThis paperPCR primers5’-gcgttctatggtagccttcag-3’
Sequence-based reagentplin1_reverseThis paperPCR primers5’-gcgtccggatagaaagctg-3’
Sequence-based reagentplin2_forwardThis paperPCR primers5’-gcagaatggcaagagttctga-3’
Sequence-based reagentplin2_reverseThis paperPCR primers5’-actgtgtgtaggactggatcctc-3’
Sequence-based reagentRpl1_forwardThis paperPCR primers5’-tccaccttgaagaagggcta-3’
Sequence-based reagentRpl1_reverseThis paperPCR primers5’-ttgcggatctcctcagactt-3’
Peptide, recombinant proteinTrehalaseSigma AldrichT8778
Peptide, recombinant proteinβ-AmyloglucosidaseSigma Aldrich10115
Antibodyanti-phospho(Ser505)-AKTCell Signal Technology (CST)Cat# 4054; RRID:AB_331414WB (1:1000)
Antibodyanti-AKTCell Signal Technology (CST)Cat# 4691; RRID:AB_915783WB (1:1000)
Antibodyanti-phospho(Thr172)-AMPKαCell Signal Technology (CST)Cat# 2535; RRID:AB_331250WB (1:1000)
Antibodyanti-phospho(Thr389)-p70 S6 kinaseCell Signal Technology (CST)Cat# 9206; RRID:AB_2285392WB (1:1000)
Antibodyanti-GFPCell Signal Technology (CST)Cat# 2956; RRID:AB_1196615WB (1:1000)
Antibodyanti-phospho-p44/42 MAPK (Erk1/2)Cell Signal Technology (CST)Cat# 4370; RRID:AB_2315112WB (1:1000)
Antibodyanti-α-tubulinDevelopmental Studies Hybridoma Bank)Clone 12G10; RRID:AB_1157911WB (1:3000)
AntibodyHRP anti-rabbit IgGCell Signal Technology (CST)Cat# 7074; RRID:AB_2099233WB (1:5000)
AntibodyHRP anti-mouse IgGCell Signal Technology (CST)Cat# 7076; RRID:AB_330924WB (1:5000)
Commercial assay or kitFirst Strand cDNA Synthesis KitThermo ScientificK1622
Commercial assay or kitSensimix SYBR Green no-ROXBiolineQT650-05
Chemical compound, drugBromophenol blueSigma AldrichSML1656
Chemical compound, drugXylene cyanolCarl RothA513.1
Chemical compound, drugBrilliant Blue FCFSigma Aldrich80717
OtherHCS Lipid Tox Deep RedThermo FisherH34477IF (1:200)
OtherAlexa Fluor 488 PhalloidinThermo FisherA12379IF (1:20)
OtherFluoromount-Gebioscience00-4958-02
OtherTRIzolInvitrogenAM9738
OtherFixable Viability Dye 780ebioscience65-0865-18FC (1:1000)
OtherSupersignal West Pico Chemiluminescent SubstrateThermo Scientific34077
OtherSupersignal West Femto Chemiluminescent SubstrateThermo Scientific34094
OtherGlucose ReagentSentinel Diagnostics17630H
Software, algorithmImageJImageJRRID:SCR_003070
Software, algorithmGraphPad PrismGraphPadRRID:SCR_002798
Software, algorithmFlowJoFlowJoRRID:SCR_008520)

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  1. Katrin Kierdorf
  2. Fabian Hersperger
  3. Jessica Sharrock
  4. Crystal M Vincent
  5. Pinar Ustaoglu
  6. Jiawen Dou
  7. Attila Gyoergy
  8. Olaf Groß
  9. Daria E Siekhaus
  10. Marc S Dionne
(2020)
Muscle function and homeostasis require cytokine inhibition of AKT activity in Drosophila
eLife 9:e51595.
https://doi.org/10.7554/eLife.51595