Iwr 1 endo

To generate stable cell lines, 46C cells seeded on 100 mm plates and
were transfected with 24 μg of linearized
pCIG-NOTCH2NL^Sh,T197I-ires-GFP or empty pCIG-ires-GFP vector,
using lipofectamine 2000 (Thermofisher). After 36 hours, GFP-positive cells
were sorted using a FACSAria III (BD Biosciences) and recovered for further
culturing. After 4 passages sorting was repeated and GFP-positive cells that
had stably integrated the plasmid DNA in their genome were recovered for
expansion and further culturing. We verified continued stable expression of
NOTCH2NL^Sh,T197I-ires-GFP or empty vector (Supplemental Figure S4). Mouse
46C ESC organoid differentiation was performed as described previously
(Eiraku et al., 2008 (link)). Briefly,
cells were seeded in ultra low attachment U-shaped 96 wells plates (Corning)
at 6000 cells per well. Cells were in mouse ESC medium without LIF and
supplemented with 3 μM IWR-1-Endo (Sigma) and 10 μM SB431542
(Sigma). Medium was replaced every other day. At day 7, medium was changed
to Neurobasal/N2 medium. Three pools of 16 organoids were isolated in TRIzol
after 6 days of differentiation for EV and NOTCH2NL^Sh,T197organoids.

To investigate the role of Wnt signaling in heart regeneration, the Wnt antagonist IWR-1-endo (Sigma-Aldrich) was used^{66 (link),67 (link)}. IWR-1 was dissolved in DMSO to prepare a 10 mM stock solution. Wild-type fish were injected intraperitoneally with 25μl of 10μM IWR-1 in PBS or DMSO (0.1% in PBS)^{68 (link)}. The injection was performed at 1 dpi and 2 dpi for the 3 dpi analysis, and for later time points, once every two days from 2 dpi until the day of sacrifice.

1×10⁴ cells were plated per well in 96-well round bottom polystyrene dishes and allowed to adhere for 16 hours before treatment with vehicle or drug. IWR-1-endo (Calbiochem, San Diego, CA, USA) or verapamil (Sigma-Aldrich, St. Louis, MO, USA) were added to the wells for 16 hours (IWR-1-endo) or 15 minutes (verapamil). After treatment, Calcein AM (Thermo-Fisher, Waltham, MA, USA) was reconstituted in DPBS and added to each well to reach a final dilution of 1:3000 (333nM), and incubated at 37ºC for 30 minutes. For doxorubicin retention assays, following treatment with IWR-1, doxorubicin was added to each well to reach a final concentration of 10µM, and incubated at 37ºC for 2 hours. Solutions were then removed, wells were rinsed with DPBS, and total fluorescent emission in each well was measured with a TECAN Infinite F200 Pro plate reader (Tecan, Zurich, Switzerland).