Teto gata4

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.

All plasmids were constructed as previously described [5] (link) and can be found on Addgene using the following catalog numbers: Troponin T-GCaMP5-Zeo (46027), tetO-Hand2 (46028), tetO-NKX2.5 (46029), tetO-GATA4 (46030), tetO-MEF2C (46031), tetO-TBX5 (46032). Plasmids that were used from Addgene also include: FUdeltaGW-rtTA (19780), psPAX2 (12260), pMD2.G (12259), and PGK-H2B-mCherry (21217). All plasmids were amplified in STBL3 bacteria (Invitrogen) and prepared with Qiagen MidiPrep Kits.