The effect of TOR inhibition on cell proliferation in <i>Nematostella </i>(Fig. 5)

Juvenile polyps were starved for 4 days and then fed during treatment with either 4uM Rapamycin (0.2% DMSO) or 0.2% DMSO for control. Before treatment (T0) and after 4 days of treatment, polyps were incubated for 30min in EdU (300uM, 2%DMSO) and dissociated using Trypsin and fixed with Formaldehyde. Cells were stained with 1μg/ml FxCycle violet DNA dye (Invitrogen) and EdU was labelled with Alexa488 using a Click-it reaction.Flow cytometry was performed a BD LSRFortessa (BD Life Sciences) and the EdU positive cells in samples were determined based on Alexa488 fluorescence in comparison with the DMSO controls on cells within the cell cycle (defined by DNA stain) using FlowJoV10.8 (BD Life Sciences)..wsp files contain the samples, gating strategy and cell populations used for the analysis in FlowJo.fcs files represent the individual flow cytometry output files that were analysed in the .wsp file