Epigallocatechin gallate (egcg)

iPSC cardiomyocytes at day 6 post initial plating were transfected with 100 ng plasmid DNA using ViaFect transfection reagent (E4981, Promega) and the following human DYRK1A and SRSF6 expression constructs: DYRK1A human clone (NM_001396, SC314641, Origene), DYRK1A human tagged ORF Clone (NM_001396, RG212584, Origene), and SRSF6 cDNA ORF clone N-HA tag (HG19052-NY, Sinobiological). Control cultures were transfected with ~ 100 ng of empty PCMV6XL5 vector (Origene).
Drug treatments were initiated 24 h post-transfection with expression constructs. Daunorubicin (DAU, 14159, Cayman Chemical) and epigallocatechin gallate (EGCG, 709035, Cayman Chemical) were added to culture media for a total incubation time of 14 h. DMSO vehicle was added to controls. After treatments with DAU, iPSC cardiomyocytes were washed with PBS, and incubated in fresh culture medium, or medium supplemented with EGCG for 24 h. Cell viability was determined with the CellTiter-Glo luminescent viability kit (G7570, Promega).

To evaluate the inhibitory activity on DPP4, the DPPIV-Glo™ Protease Assay (Promega, USA) was used to assess the effect of EGCG on DPP4 enzyme activity in vitro. At the same time, the inhibitory effect of other compounds present in tea such as epigallocatechin, epicatechin gallate, epicatechin, and catechin on DPP4 activity was also determined. DPP4 enzyme and EGCG were purchased from Sigma. Sitagliptin (Sigma, USA), a DPP4 inhibitor [29 (link)], was used as a reference compound. Epigallocatechin, epicatechin gallate, epicatechin, and catechin were obtained from bidepharm (Shanghai, China). Half maximal inhibitory concentration (IC50) values were determined by nonlinear regression in GraphPad Prism 6 software.

Epigallocatechin-gallate (EGCG, Sigma-Aldrich, St. Louis, MO, USA, E4143) and honokiol (HNK, Sigma-Aldrich, St. Louis, MO, USA, H4914) were dissolved in DMSO at 100 mM and 10 mM, respectively, and stored at −20 °C. To determine cell viability, EGCG and HNK were serially diluted onto 1 × 10⁴ A549 cells for 26 h. CellTitre-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) reagent was then added to cells following manufacturer’s instructions. Values were normalised to DMSO controls. For infection assays, A549 cells were treated with EGCG or HNK 2 h prior to and/or throughout a 24 h infection with ZIKV-Nanoluc (multiplicity of infection (MOI) 0.1). Where specified, ZIKV-Nanoluc was incubated with either EGCG or DMSO for 2 h in DMEM plus 2% FBS prior to infection of cells. To measure activity of Nanoluc expressed by a virus, cells were first lysed in Passive Lysis Buffer (Promega, Madison, WI, USA) before detection with Nano-Glo Luciferase Assay System plate reader (Promega, Madison, WI, USA). Values were normalised to infected cells treated with DMSO only. All drug treatments were performed in DMEM supplemented with 2% FBS at 37 °C with 5% CO₂.