1. Transferrin 1 Functions in Iron Trafficking and Genetically Interacts with Ferritin in Drosophila melanogaster 
Guiran Xiao, Zhi-Hua Liu, Mengran Zhao, Hui-Li Wang, Bing Zhou 
Cell Reports 
Volume 26, Issue 3, Pages e5 (January 2019)
DOI: /j.celrep
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2. Cell Reports 2019 26, 748-758.e5DOI: (10.1016/j.celrep.2018.12.053)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1 Tsf1 Is Involved in Iron Homeostasis and Is Expressed Broadly
(A) RT-PCR analysis of Tsf1 mRNA abundances in third-instar larvae, with rp49 as internal control (a). Western blot showing Tsf1 expression in third-instar larvae (b). β-actin was used as the loading control; approximate molecular weights of marker proteins are indicated. Genotypes used in (a) and (b) are da-GAL4 >w1118 (control), da-GAL4>Tsf1-RNAi, or da-GAL4>UAS-Tsf1-OE. n = 15 larvae per group.
(B) Tsf1-RNAi flies displayed iron-sensitive defects. Progeny were reared on food supplemented with iron (5 mM ferric ammonium citrate [FAC]), zinc (2 mM ZnCl2), iron chelator (200 μM BPS) and zinc chelator (25 μM N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine [TPEN]). Genotypes used are Act-GAL4 > w1118 (control), Act-GAL4>Tsf1-RNAi, or Act-GAL4>UAS-Tsf1-OE. n = 80–120 flies per vial, n = 6 vials per experimental group.
(C) qPCR analysis of change of whole-body Tsf1 mRNA abundance after Tsf1 knockdown in different tissues, such as the midgut (NP3084), the fat body (Cg-GAL4), the trachea (104879), and the CNS (elav-GAL4). rp49 was used as the loading control. n = 15 larvae per group.
(D) Western blot of Tsf1 abundance after Tsf1 knockdown in different tissues. β-actin was used as the loading control. n = 15 larvae per group.
(E) Quantitative measurement of (D). Both the mRNA and the protein levels of Tsf1 are dramatically suppressed in da-GAL4>Tsf1-RNAi, Cg-GAL4>Tsf1-RNAi, and >Tsf1-RNAi larvae, whereas they are slightly reduced in NP3084>Tsf1-RNAi and elav-GAL4>Tsf1-RNAi. n = 15 larvae per group.
(F) Eclosion rates of da-GAL4>Tsf1-RNAi and Cg-GAL4>Tsf1-RNAi were reduced, whereas no significant difference was observed for Tsf1 knockdown in other tissues. n = 70 larvae per vial, n = 6 vials per experimental group.
(G) qPCR analysis of Tsf1, Fer1HCH, and Fer2LCH of w1118 third-instar larvae in response to iron changes. rp49 was used as the loading control. n = 15 larvae per group.
(H) Western blot of Tsf1, Fer1HCH, and Fer2LCH of w1118 third-instar larvae in response to iron changes. β-actin was used as the loading control. n = 15 larvae per group.
All values are presented as mean ± SEM of the biological replicates; n ≥ 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, two-tailed Student’s t test. OE, overexpression. See also Figure S1.
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4. Figure 2
Fat Body Tsf1 Knockdown Leads to Iron Deficiency in the Fat Body but Iron Accumulation in the Gut
(A) Ferrozine assays indicated a significant increase of iron content in the gut and a decrease of iron content in the fat body of Cg-GAL4>Tsf1-RNAi third-instar larvae. n = 40 guts or fat bodies per group.
(B) Aconitase activity suggested iron elevation in the gut and iron deficiency in the fat body of Cg-GAL4>Tsf1-RNAi third-instar larvae. n = 40 guts or fat bodies per group.
(C) Staining of ferric iron (bound to ferritin) in the 1 mM FAC-fed third-instar larval gut. The anterior midgut (red arrows) and posterior midgut (green arrows) of Cg-GAL4>Tsf1-RNAi larvae deposited a higher amount of iron than the control. Shown are images in bright fields. n = 6–10 replicates per group.
(D) Staining of ferric iron on native PAGE. β-actin was used as the loading control. n = 40 guts or fat bodies per group.
(E) Western blot analysis showing that Tsf1 knockdown in the fat body (Cg-GAL4>Tsf1-RNAi) resulted in an increased ferritin level in the gut and a decreased ferritin level in the fat body. β-actin was used as the loading control. n = 40 guts or fat bodies per group.
Genotypes are Cg-GAL4 > w1118 (control) and Cg-GAL4>Tsf1-RNAi. All values are presented as mean ± SEM of the biological replicates; n ≥ 3. ∗p < 0.05, ∗∗p < 0.01, two-tailed Student’s t test. See also Figure S1.
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5. Figure 3 Tsf1 Made in the Fat Body Could Be Found on the Gut Surface
(A) Tsf1-EGFP synthesized in the fat body (red arrows) could be transferred to hemolymph (green arrows) and other tissues, including the gut (blue arrows). Scale bars, 500 μm. Genotypes are Cg-GAL4>EGFP (control) and Cg-GAL4 > Tsf1-EGFP. n = 6 replicates per group.
(B) Western blot analysis showing that Tsf1 knockdown and overexpression in the fat body resulted in corresponding change of Tsf1 levels in the gut. β-actin was used as the loading control. Genotypes are Cg-GAL4 > w1118 (control), Cg-GAL4>Tsf1-RNAi, and Cg-GAL4 > Tsf1-OE. n = 40 guts or fat bodies per group.
(C) Immunohistochemistry indicated that Tsf1-EGFP synthesized in the fat body could be transferred to the gut surface. Scale bars, 100 μm. Genotypes are Cg-GAL4>UAS-sig-EGFP (control) and Cg-GAL4 > UAS-Tsf1-EGFP. The sig-EGFP contains a secretion signal of Fer1HCH fused to the N-terminal of EGFP, making it secreted. n = 6–10 replicates per group.
(D) Magnified view of the regions highlighted in (C).
n ≥ 3. See also Figure S2.
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6. Figure 4
Tsf1 Made in the Gut Could Be Found in the Hemolymph and Involved in Iron Metabolism
(A) When EGFP is driven by NP3084, the signal is concentrated in the intestine (red arrows) and salivary gland. When Tsf1-EGFP is driven by NP3084, the signal is diffused and hard to be detected in the gut (except salivary gland). The blue arrows indicate the cuticles. The living larvae were captured with 1×Microscope Objective Lens in bright and dark fields, and the final images were merged together from cuticle and GFP images in Adobe Photoshop CS6. Scale bars, 500 μm. n = 6 replicates per group.
(B) Western blot analysis showing that Tsf1-EGFP synthesized in the gut was found in the hemolymph. n = 40 larvae.
(C) Western blot analysis showing that Tsf1-EGFP synthesized in the gut was found in the body parts other than the gut. n = 15 larvae or 40 guts or 40 whole body minus guts per group.
(D) Midgut-specific Tsf1 RNAi flies were specifically sensitive to iron overdose.
Genotypes used in (A)–(C) are NP3084>UAS-EGFP (control) and NP3084 > UAS-Tsf1-EGFP. Genotypes used in (D) were NP3084 > w1118 (control) and NP3084>Tsf1-RNAi. n = 20 flies per vial, n = 10 vials per group. n ≥ 3.
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7. Figure 5
Tsf1 Knockdown in the Gut Rescues Ferritin and ZIP13 RNAi Phenotypes
(A) Decreased eclosion rate of midgut-specific Fer1HCH-RNAi was rescued by Tsf1 knockdown while exacerbated by Tsf1-OE. Midgut-specific dZIP13-RNAi flies are sensitive to iron depletion in food. Tsf1 knockdown in the gut rescued the phenotype, while Tsf1-OE exacerbated it. n = 70 larvae per vial, n = 6 vials per experimental group.
(B) Decreased iron contents caused by dZIP13-RNAi or Fer1HCH-RNAi in the midgut were rescued by Tsf1 RNAi. n = 40 larvae per group.
(C) Induced ferritin expression caused by dZIP13-RNAi in the midgut was rescued by Tsf1 RNAi. n = 40 guts per group.
(D) Staining of ferric iron in the gut of larvae cultured on 1 mM FAC. Shown are images in bright fields. Scale bars, 500 μm. Genotypes used were NP3084 > w1118 (control), NP3084 > dZIP13-RNAi, NP3084 > dZIP13-RNAi;Tsf1-RNAi, NP3084 > Fer1HCH-RNAi, and NP3084 > Fer1HCH-RNAi;Tsf1-RNAi. n = 6–10 replicates per group.
(E) Ectopically induced expression of Fer1HCHG188 in dZIP13-RNAi was restored to normality by Tsf1 knockdown. Shown are representative images in bright and dark fields. Scale bars, 500 μm. Genotypes used were NP3084 > w1118 (control), NP3084 > dZIP13-RNAi, NP3084 > dZIP13-RNAi;Tsf1-RNAi, and NP3084 > dZIP13-RNAi;Tsf1-OE. n = 6–10 replicates per group.
Results are presented as mean ± SEM of the biological replicates; n ≥ 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, two-tailed Student’s t test. OE, overexpression. See also Figure S3.
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8. Figure 6
Tsf1 and Ferritin Display Strong Functional Antagonism in the Fat Body and Midgut, but Not the Nervous System or Eyes
(A) Retarded growth and lethality of ubiquitous or midgut- or fat body-specific Fer1HCH knockdown could be dramatically rescued by Tsf1 knockdown. The 3rd instar larval stage, pupal stage and adulthood (imago) were shown. n = 6 replicates per group.
(B) Decreased eclosion rate of Fer1HCH-RNAi driven by da-GAL4 was rescued by Tsf1 knockdown. n = 70 larvae per vial, n = 6 vials per group.
(C) Decreased eclosion rate of midgut-specific Fer1HCH-RNAi was rescued by Tsf1 knockdown. n = 70 larvae per vial, n = 6 vials per group.
(D) Decreased eclosion rate of fat body-specific Fer1HCH-RNAi was rescued by Tsf1 knockdown. n = 70 larvae per vial, n = 6 vials per group.
(E) Tsf1 knockdown had no effect on the retarded growth or reduced survival of elav-GAL4 > Fer1HCH-RNAi. n = 70 larvae per vial, n = 6 vials per group.
(F) Tsf1 knockdown had no effect on the abnormal compound eye morphology of elav-GAL4 > Fer1HCH-RNAi or GMR-GAL4 > Fer1HCH-RNAi. n = 6 replicates per group.
All values are presented as mean ± SEM of the biological replicates; n ≥ 3. ∗∗p < 0.01, ∗∗∗p < 0.001, two-tailed Student’s t test. OE, overexpression.
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9. Figure 7
Model to Explain the Function of Drosophila Tsf1 in Iron Metabolism
Tsf1 made in the gut excretes through the secretion pathway, wherein it competes with ferritin for iron (brown circle). This competition becomes especially consequential when dZIP13 is limited. Tsf1 made in the fat body circulates in the body and, in a way somewhat similar to that of mammalian TF, accepts iron from the gut surface and traffics to the fat body. See the text for details.
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