M3 media

Drosophila S2R + cells express all of the Wg signaling components necessary to respond to exogenously added Wg^{36 (link)}. For Arm, UAS-ArmAR2 and USA-HA control transfections, 1 µg DNA, 1 µg WISER (luciferase reporter plasmid) (gift from J.P Vincent) and 1 µg act-Gal4 (in the well of UAS-HA) in 98 µl EC buffer were combined with 16 µl Enhancer (Qiagen), 38 µl Effectene (Qiagen) in 3.5 × 10⁶ cells/1.6 mL of growth media in a 6-well plate. To assess the effect of Arm and Arm AR2 on Wg signaling, 400 µl of media with or without Wg protein was added 2 days after transfection. 1 day later, the cells were lysed to assess luciferase levels using the Dual-Luciferase Reporter Assay System (Promega) containing Tcf reporter (firefly luciferase) and a constitutively active control reporter (renilla luciferase). Wg activity was calculated as the ratio of firefly to renilla luciferase activities with SEM (Standard error of the mean) of three independent experiments with the HA control. Statistical significance was based on a two-tailed t test. To obtain Wg-containing media, S2 tub-wg cells (DGRC) and S2 cells were grown in M3 media (Sigma) as described^{37 (link)}. Cells were pelleted by centrifugation. The presence of Wg protein was confirmed by Western blotting by mouse anti-Wg antibody (DSHB).

Drosophila S2 cells were cultured in M3 media (Sigma) with 10% insect medium supplement (Sigma). Transfection was carried out with Effectene reagent (Qiagen, Hilden, Germany) according to manufacturer’s instructions.
HEK293 cells were cultured in Dulbecco’s modified Eagles medium (DMEM) with 10% fetal bovine serum (FBS). For siRNA-mediated gene silencing, siRNA was transfected into HEK293 cells by reverse transfection using Lipofectamin 2000 to a final concentration of 10 μM. siRNAs were generated from Bioneer (Korea). siRNA sequences are shown in Table S1. Total RNA of siRNA-transfected cells was extracted 48 hours after transfection by Qiazol (Qiagen, UAS) according to the manufacturer’s instruction. For plasmid transfection, pCEP4 SMAD2 (Addgene, #16484) and tetO-GATA4 (Addgene, #46030) were used. cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Realtime PCR Master Mix (Toyobo, Japan) was used to perform qRT-PCR assays. qRT-PCR primer sequences are shown in Table S2.
ACE vector (Sino Biological, HG11598-UT) was used for overexpression experiment and western blot analysis, and Angiotensin II (Sigma-Aldrich, A9525) was treated for western blot analysis and cell viability assay. For western blot analysis, rabbit anti-ERK (Cell Signaling, 4595 T) and rabbit anti-phospho ERK (Cell Signaling, 3101 S) were used.

Drosophila S2 cells were grown in M3 media (Sigma-Aldrich) supplemented with 10% IMS (Sigma-Aldrich) at 25 °C. Transfections were carried out with transfection reagents effectene (Qiagen) or cellfectin (Invitrogen) according to the manufacturers’ instructions. For each transfection, a total of 1–2 μg DNA was used.
To establish S2 sona-HA cell line that stably expresses sona-HA, hygromycin B selection system was used according to the manufacturers’ instructions (Invitrogen life technologies). Briefly, 4 × 10⁶S2 cells were cotransfected with total 1 μg of two plasmids, pAC sona-HA and pCoHygro (19:1 ratio), for 3 days. Then, the culture medium was changed to selective medium containing 150 μg/ml of hygromycin B (Invitrogen). The selective medium was replaced every 5 days, and the sona-HA cell lines were established after 3 weeks. The established sona-HA cell line was maintained in selective medium containing hygromycin B.

Drosophila S2 cells were cultured in M3 media (Sigma, Saint Louis, Missouri) with 10% Fetal Bovine Serum (Thermo, Waltham, MA). S2 cells were transfected using Effectene (Qiagen, Venlo, Netherlands) or X-tremeGENE HP DNA transfection reagent (Roche, Basel, Switzerland) according to the manufacturer’s manual. pAc5.1 empty vector was used to transfect with an equal amount of plasmids. A total of 1–3 μg DNA was used for each transfection. For co-IP, transfected S2 cells were incubated during 2~3 day for producing proteins from transfected DNA.

Live pupae with partially ripped cuticle were incubated in 10μg/ml FM4-64 (Life Technologies) in M3 media (Sigma-Aldrich) for 15-30m at 25°C. After the incubation, wings were directly subjected to confocal microscopy.

S2R + cells were grown in M3 media (Sigma) supplemented with 10% 10X insect medium supplement (Sigma) and 1% penicillin–streptomycin solution. Cultured cells were transfected with 1–3 µg DNA using Cellfectin II Reagent following a protocol from the manufacturer (Thermo Fisher Scientific).