PluriSIn 1 (NSC 14613) is an inhibitor of stearoyl-CoA desaturase (SCD1), the key enzyme in oleic acid biosynthesis, revealing a unique role for lipid metabolism in hPSCs.

SCD1 activity assays: Cells are plated in 6-well plates at a density of 50k to 100k cells per well. 24 h later, 20 μM PluriSIn #1 or 0.2% DMSO-control are added to the cells. After 12 h of incubation at 37 ℃, 5% CO2, the old medium is removed, cells are washed with PBS, and new medium containing 2.3 μM of 0.75 UCi [1-14C] Stearic Acid is added. The cells are incubated for up to 4 h at 37 ℃, 5% CO2. After the incubation period, the medium is discarded and the cells are washed 3 times with 2 mL of PBS. 2 mL of the mixture n-hexane: isopropanol (3:2 v:v) are added, and the cells are incubated for 30 min at 37 ℃, 5% CO2. 2 mL Folch solution (chloroform: methanol,2:1,v:v) are subsequently added. The liquid is transferred to tubes for phase partition by adding 1 mL water. The lower organic phase is evaporated and used for lipid saponification and TLC separation of the free [1-14C] Stearic Acid (substrate) and [1-14C] Oleic Acid (formed product). Lipids extracted from the cells are applied to TLC plates previously immersed in 10% NO3 Ag and activated at 120℃x60 min. Unlabeled stearic and oleic acid are added to each application point as carriers and as internal standards for identification. The plates are run with a solvent mixture of Chloroform:MeOH:AcH:DDW (90:8:1:0.8). The free fatty acids are detected by U.V. after spraying the TLC with a 2',7',dichlorofluorescein solution. The spots corresponding to stearic and oleic acid are scraped and the radioactivity counted in a sc intillating counter. SCD1 desaturase activity is calculated from the percent conversion of substrate to product and the conversion to pmol/min/106 cells.

Relative cell numbers are determined by fixating the cells with 0.5% glutardialdehyde and staining with methylene blue dissolved in 0.1 M boric acid (pH 8.5). Color extraction is performed using 0.1 M hydrochloric acid, and the staining (which is proportional to cell number) is quantitated by measuring absorbance at 650 nM.(Only for Reference)