Fig. 1. Potential ubiquitin-specific protease (USP) candidates regulating H2B monoubiquitination. (A) The effect of CRISPR/Cas9-mediated knockout (KO) of each USP candidate (Usp7, Usp12, Usp21, and Usp37) on the expression level of histone H2B monoubiquitination (H2Bub1) was estimated by Western blotting. (B) The expression of H2Bub1 was graphically represented. *p<0.05, ***p<0.001 by paired t-test. (C) Schematic representation of retinoic acid (RA)-induced P19 cell neuronal differentiation schedule. P19 cells formed aggregates for four days with 10 μM of RA. Aggregates were collected and seeded in cell culture plates on day 4 and harvested at the indicated time points. (D) RA-induced neuronal differentiation of P19 cells at different days. Samples were collected at the indicated time points and the expressions of Oct3/4 and βIII-tubulin protein were analyzed by Western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was analyzed as a loading control. (E) Fluorescence microscopy of P19 cell aggregates after transfection of Cas9 tagged with red fluorescent protein (RFP) along with single guide RNA (sgRNA) targeting Usp7. Scale bar=100 μm.