Green fluorescent protein (GFP) Assay

miPSCs cells were seeded at a density of 2 × 10³ cells/well in 96-well black plates (Corning Inc., NY) using miPS medium overnight. On the second day, half of the medium was replaced with a fresh medium mixed with compounds every day, up to day six. On day seven, the cells were washed twice with 1× PBS and lysed in a 50 μl/well lysis buffer followed by the incubation at 37 °C for 5mins. The relative fluorescence intensity was then measured at 509 nm, excited at 488nm, using a microplate reader (SH-9000lab, Corona, Japan). The read of each well was obtained from 9 points at a distance of 1 mm from the bottom of each well flashed 30 times.

The GFP fluorescence from the miPSCs cultured in CM without signal inhibitors was used as a control to determine the threshold of the bottom line in order to distinguish the effect of the signal inhibitors. The fluorescence from 6 wells was measured 5 times from independent experiments, the averages and standard deviations of the readings were calculated (Table ^S1). Next, we assessed the effect of the inhibitors in concentrations between 0 and 10 μM by the intensity of the GFP fluorescence and determined the optimal concentration which enhanced the fluorescence of GFP (Table ^S2).