Fig 2: Identification of a peptide agonist of JMJD1C. (A) Stapled peptides of the JMJD1C zinc finger and jumonji domains. a,a-Disubstituted non-natural amino acids (X) containing olefinic side chains of varying lengths were synthesized. Non-natural amino acid substitutions were made to flank three (substitution positions i and i + 4) amino acids within the JMJD1C zinc finger and jumonji domains, so that the reactive olefinic residues would reside on the same face of the a-helix. ZFD, zinc finger domain of JMJD1C; TAT, HIV TAT sequence; X = S4; N-myr, myristoylation at the N-terminal of the peptide. (B–H) The effect of the JMJD1C-originated peptides on the cell proliferation. Cell lines MV4-11, SEM, HL-60, Jurkat, and NK-92 were maintained, as described in the Materials and Methods. Cells were seeded at 30,000/mL in 100 µL medium in V-bottom 96-well plates and incubated with the indicated peptides for the duration shown. Cells were measured for the cell proliferation using an ATP detection kit, as described in the Materials and Methods. Three replicated experiments were performed, and a p value smaller than 0.05 was regarded as statistically significant. (I,J) The effect of the JMJD1C-derived peptide SAH-JZ3 on JMJD1C (I) and the SCD (J) protein expression. Cell lines MOLM-13 and THP-1 were maintained, as described in the Materials and Methods. Cells were incubated with the indicated peptides for 3 d and then collected for western blotting. Three replicated experiments were performed. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The intensity ratio of each band was calculated by normalizing the control to 1.