Figure 4.

Crotonic acid promotes pluripotency and enhances proteasomeactivity

A. Relative expression of pluripotency and differentiation genes measured by RT-qPCR in ESCs cultured in S/L with or without 10 mM crotonic acid. Data are presented as mean ± SEM (n = 3 biological replicates with 3 technical replicates each). B. Relative expression of representative pluripotency and differentiation genes measured by RT-qPCR in ESCs cultured in S/L with or without 10 mM crotonic acid and subjected to differentiation by withdrawing LIF over a time-course of 4 days. Data are presented as mean ± SEM (n = 3 biological replicates with 3 technical replicates each). C. Schematic representation of the proteasome complex and the components identified in our crotonylome. D. Chymotrypsin-like proteasome activity measurement in ESCs cultured in S/L with or without 10 mM crotonic acid treatment for 48 h (n = 4 biological replicates). E. Chymotrypsin-like proteasome activity measurement in pre-purified proteasome by native gel electrophoresis. ESCs were cultured in S/L with or without 10 mM crotonic acid treatment for 48 h. Samples were resolved by denatured SDS-PAGE and Western blotting for analyzing the 20S proteasome level. ACTIN was used as a loading control. F. Quantified densitometry of the result shown in (E). Data are presented as mean ± SEM (n = 4 biological replicates). G. Representative Western blotting of lysine crotonylation, poly-ubiquitination, and K48-ubiquitination in ESCs cultured in S/L and untreated or treated with different concentrations of crotonic acid for 48 h. ACTIN was the loading control. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant (two-tailed unpaired Student’s t-test).