Journal: bioRxiv

Article Title: Functional characterization of eicosanoid signaling in Drosophila development

doi: 10.1101/2025.01.13.632770

Figure Lengend Snippet: (A) Unrooted maximum-likelihood phylogenetic tree of class A G protein-coupled receptors (GPCRs) in H. sapiens and D. melanogaster . GPCRs in D. melanogaster are indicated by blue branches, whereas branches for H. sapiens GPCRs are color-coded based on different classes of ligands. The scale bar indicates an evolutionary distance of 0.5 amino acid substitutions per site. Opsins and leucine-rich repeat containing GPCRs are not included in the phylogenetic tree. Accession numbers of the receptors analyzed are listed in S1 Table. (B) List of lipid mediator receptors in H. sapiens and their Drosophila orthologs. (C) Luminescence responses of S2 cells co-expressing Aequorin and G α 15 , along with CG7497/PGR or H. sapiens PGE2 receptor EP2 ( HsEP2 ), to prostanoids or their synthetic analogs. Relative luminescence normalized by luminescence in ligand-free controls is shown. Cells expressing CG7497/PGR were significantly activated by all ligands, whereas cells expressing EP2 were activated only by PGE2. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001 (Dunnett’s test vs mock treatment control).

Article Snippet: Early pupae (up to 24 h APF) were collected and kept in Drosophila narrow vials (Genesee Scientific) containing wet tissue at 25°C under 12 h-light and 12 h-dark photoperiod.

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Functional characterization of eicosanoid signaling in Drosophila development

doi: 10.1101/2025.01.13.632770

Figure Lengend Snippet: (A) Schematic diagram of prostanoid synthetic pathways. Multiple prostanoids are synthesized from arachidonic acid (AA) by different enzymes. (B) Drosophila enzymes orthologous to human prostanoid synthases were identified by phylogenetic analyses. Phylogenetic trees are shown in S4–S10 Figs. PGD, PGE, and PGF synthases are highly conserved in insects, although D. melanogaster lacks some of them. (C) Prostanoids detected in whole body extracts of pupae by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomics. AA was detected in intact pupae at 0–4 hours after pupation, whereas prostanoids were undetectable. PGD2, PGE2, and PGF2α were detected in pupae that were raised in AA-supplemented diet during larval development. Data shown are mean total amount of eicosanoids in the whole body per animal ± SEM. n = 4–5.

Article Snippet: Early pupae (up to 24 h APF) were collected and kept in Drosophila narrow vials (Genesee Scientific) containing wet tissue at 25°C under 12 h-light and 12 h-dark photoperiod.

Techniques: Synthesized, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Functional characterization of eicosanoid signaling in Drosophila development

doi: 10.1101/2025.01.13.632770

Figure Lengend Snippet: (A) Developmental phenotype of PGD and PGE synthase ortholog mutants. Null mutant of cPges / p23 and triple mutants of Mgst - , CG33177 , and CG33178 died during pupa-adult development. Multiple comparison Chi-square test with Bonferroni correction was applied among cPges / p23 mutants including the heterozygous control. *** p < 0.001. (B) Representative images of cPges / p23 knockout flies. About 70% of flies died before wing pigmentation, whereas ∼30% of flies died as pharate adults. Scale bar, 200 µm. (C, D) Relative expression levels of hypoxia response genes, Ldh (C) and Hph (D), in the PGD and PGE synthase ortholog mutants. The cPges / p23 mutant showed high expression of hypoxia response genes at 24 and 48 hours after puparium formation (APF). Expression levels are normalized by the levels of a reference gene, rp49 , in the same cDNA samples and shown as relative to 0 hours APF of GstS1 - . n = 3–4. p < 0.001 (Tukey’s test). (E) Rescue of the cPges / p23 mutant by Gal4 -driven expression of either cPges / p23 or human prostaglandin E synthase 3 ( HsPTGES3 ). The lethal phenotype was rescued by overexpressing cPges / p23 or HsPTGES3 either ubiquitously ( tubP-Gal4 ), in PGR expression sites ( PGR-Gal4 ), in tracheal cells ( btl-Gal4 ), or in the epidermis ( Eip71CD-Gal4 ). ** p < 0.01, *** p < 0.001 (Chi-square test vs + > cPges / p23 or + > HsPTGES3 with Bonferroni correction). (F) Developmental deficiency caused by cPges / p23 RNAi. cPges / p23 knockdown flies died during pupa-adult development when induced using either a ubiquitous driver (tubP-Gal4 ) or a combination of the tracheal cell ( btl-Gal4 ) and epidermis ( Eip71CD-Gal4 ) drivers. Lethality caused by cPges / p23 RNAi in tracheal cells and epidermis was significantly rescued by 40% O 2 . *** p < 0.001 (multiple comparison Chi-square test vs + > cPges / p23 RNAi with Bonferroni correction, or 20% O 2 vs 40% O 2 ). (G) PGE2 conversion assay using Drosophila S2 cells. Arachidonic acid (AA) was added to the culture medium of S2 cells transfected with cPges / p23 or HsPTGES3 along with a H. sapiens cyclooxygenase ( HsCOX1 or HsCOX2 ), and PGE2 amount in the medium was determined. The basal levels of PGE2 immunoactivity in all samples is likely due to the cross-reactivity of the antibody used in the assay. Increased PGE2 immunoreactivity was detected in cyclooxygenase -expressing cells regardless of cPges / p23 or HsPTGES3 transfection. n = 4. (H) Reduction of the PGE2 synthetic activity of S2 cells by knocking down endogenous cPges / p23 expression. AA was added to the culture medium of S2 cells transfected with HsCOX2 and treated with cPges / p23 dsRNAs. PGE2 amount in the medium was determined. n = 2 (AA 0 µM) or n = 4 (AA 10 µM). *** p < 0.001 (Dunnett’s test vs EGFP dsRNA-treated cells).

Article Snippet: Early pupae (up to 24 h APF) were collected and kept in Drosophila narrow vials (Genesee Scientific) containing wet tissue at 25°C under 12 h-light and 12 h-dark photoperiod.

Techniques: Mutagenesis, Comparison, Control, Knock-Out, Expressing, Knockdown, Transfection, Activity Assay

Journal: PLoS Genetics

Article Title: Cholesterol Metabolism Is Required for Intracellular Hedgehog Signal Transduction In Vivo

doi: 10.1371/journal.pgen.1002224

Figure Lengend Snippet: In vitro analysis also shows a decreased response to hedgehog protein in mouse embryonic fibroblasts (MEFs). Wild-type, but not mutant, MEFs show increased cell growth upon treatment with SHH (A; *p = 0.001 for wt; n = 4). Quantitative RT-PCR analysis also shows a reduced response of Gli1 (B; *p = 0.002 for wt, p = 0.01 for rud ; n = 3) and Patched (C; *p = 0.001 for wt; n = 3) upon SHH treatment in mutant MEFs. β-galactosidase accumulates in SHH-treated wild-type; Ptc-lacZ MEFs, but not rud;Ptc-lacZ MEFs (D; *p = 0.005 for wt; n = 4). (E) PzP53Med ( Ptc-lacZ;p53 medulloblastoma) cells infected and clonally selected with control plKO.1 lentivirus respond to SHH treatment with accumulation of β-galactosidase (* p = 0.07 for wt; n = 4). Two independent clonal lines infected with Hsd17b7 RNAi constructs do not respond as robustly to SHH treatment.

Article Snippet: Mouse embryonic fibroblasts were generated using standard methods and plated at a density of 20,000 cells/cm 2 in the presence or absence of 200 ng/mL SHH amino terminal peptide (R&D Systems).

Techniques: In Vitro, Mutagenesis, Quantitative RT-PCR, Infection, Control, Construct

Journal: PLoS Genetics

Article Title: Cholesterol Metabolism Is Required for Intracellular Hedgehog Signal Transduction In Vivo

doi: 10.1371/journal.pgen.1002224

Figure Lengend Snippet: Wild-type (A–I) and rudolph (J–R) mutant MEFs were transfected with a SMO-GFP plasmid and acetylated α-tubulin immunocytochemistry was used to identify the primary cilium. SMO-GFP in untreated cells is seen outside the cilium (A–C, J–L), accumulating at the base of the axoneme (not shown), or throughout the cilium (D–F, M–O). Treatment with SHH protein localizes SMO-GFP throughout the cilium in the majority of cells observed (G–I, P–R). All images were taken at the same magnification and the scale bar in C represent 10 µm.

Article Snippet: Mouse embryonic fibroblasts were generated using standard methods and plated at a density of 20,000 cells/cm 2 in the presence or absence of 200 ng/mL SHH amino terminal peptide (R&D Systems).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Immunocytochemistry