Drosophila medium

The upstream 1.744 kb regulatory region of CPLCG5 was cloned into a pGL3-basic Firefly luciferase reporter vector (Promega, Madison, WI, USA) to generate CPLCG5-pGL3-basic. The ORF of FTZ-F1 was cloned into pEGFP-N1 (EGFP, enhanced green fluorescent protein) (Solarbio, Beijing, China) to generate FTZ-F1-EGFP. Single mutation of the putative response elements was performed using a QuickMutation™ Kit (Beyotime) using the CPLCG5-pGL3-basic plasmid as a template. Mutation positions of the FTZ-F1 binding site are shown in Fig. 5a.
Drosophila S2 cells were maintained at 28 °C in Drosophila medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco). Cell transfections were conducted using the Effectene® Transfection Reagent (Qiagen, Hilden, Germany). Endotoxin-free plasmid DNA (0.2 μg of the constructs and 0.02 μg of pRL-TK) was mixed with 5 μl of Effectene®Transfection Reagent according to the manufacturer’s instructions. After 48 h of transfection, the cells were lysed and subjected to a luciferase assay performed under the Dual Luciferase Reporter Assay System (Promega).

The protein synthesis in fly brains was assessed using the Click-iT Plus OPP Alexa Fluor 594 Protein Synthesis Assay Kit (Thermo Fisher Scientific, C10457). Fly brains were dissected in Drosophila medium (Gibco, 21720024) and then incubated in a medium containing 1:1000 (20 µM) of Click-iT OPP reagent at room temperature for 30 min. The brains were washed three times with PBS, and then fixed with 4% PFA (Biosharp, BL539A) for 1 hr at room temperature. The brains were permeabilized and blocked in 5% BSA (Biofroxx, 4240GR005) in 0.3% PBST (PBS with 0.3% Triton X-100) for 90 min at room temperature, and then washed three times with PBS. The brains were incubated with primary antibodies (GFP, 1:200, Cell Signaling Technology, 2955) overnight at 4℃, washed three times with PBS, and incubated with secondary antibodies (1:200, Thermo Fisher Scientific) and DAPI (1:500, Sigma, D9542) for 1 hr at room temperature. For the Click-iT reaction, brains were incubated in the Click-iT reaction cocktail in the dark at room temperature for 30 min. Brains were then washed three times with PBS and imaged by confocal fluorescence microscopy (ZEISS LSM880).