Fig 2: JMJD1C expression in the PMA-induced differentiation of K562, HEL, and MEG-01 cells, TPO-induced differentiation of the cord blood cells, and thrombocytopenia patients. (A) JMJD1C mRNA expression in the PMA-induced differentiation of K562 cells. Cells were seeded at 2.5 × 105/mL cells and treated with 1 nM PMA for 72 h. Cells were then collected and lysed in TRIzol buffer. qPCR was performed. Three independent experiments were performed, and a p value smaller than 0.05 was regarded as statistically significant. (B,C) JMJD1C and KDM3B protein expressions in the PMA-induced differentiation of K562 cells. Cells were seeded at 2.5 × 105/mL cells and treated with 1 nM PMA for 72 h. Cells were then collected and lysed in a protein lysis buffer. WB was performed. Three independent experiments were performed. (D) Histone 3 lysine 9 mono- and di-methylation (H3K9-me1 and H3K9-me2) levels in the PMA-induced differentiation of K562 cells. Cells were treated, as described above. (E) JMJD1C mRNA expression in the PMA (10 nM)-induced differentiation of HEL cells. Cells were treated, as described above. (F,G) JMJD1C and KDM3B protein expression in the PMA (10 nM)-induced differentiation of HEL cells. Cells were treated, as described above. (H) H3K9-me1 and H3K9-me2 levels in the PMA-induced differentiation of HEL cells. Cells were treated, as described above. (I) JMJD1C mRNA expression in the PMA (10 nM)-induced differentiation of MEG-01 cells. Cells were treated, as described above. (J) JMJD1C protein expression in the PMA (10 nM)-induced differentiation of MEG-01 cells. Cells were treated, as described above. (K) JMJD1C mRNA expression in the TPO-induced differentiation of the CD34+ human cord blood cells. Human cord blood cells were obtained and CD34+ cells were isolated, as described in the Materials and Methods. An amount of 10 ng/mL was used to induce the differentiation of the CD34+ human cord blood cells, and the cells were then collected and lysed in TRIzol buffer. qPCR was performed. Three independent experiments were performed, and a p value smaller than 0.05 was regarded as statistically significant. (L) JMJD1C mRNA levels were measured in peripheral blood mononuclear cells (PBMCs) from 16 healthy subjects and four age-, race-, and sex-matched subjects with thrombocytopenia (Supplementary Table S2, p = 0.049, Mann–Whitney U test). The mean expression of JMJD1C in the normal controls and thrombocytopenia patients was 2.049 and 1.184, respectively. PBMCs were obtained, as described in the Materials and Methods, and the cells were used for the qPCR detection. NC, normal controls; TP, thrombocytopenia. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The intensity ratio of each band was calculated by normalizing the control to 1.

Fig 3: 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.

Fig 4: The effect of the JMJD1C knockdown on cell differentiation of K562 and MEG-01 cells. (A) JMJD1C knockdown in K562 cells. Cells were seeded at 2.5 × 105/mL cells and infected with lentivirus carrying JMJD1C shRNA for 72 h. Cells were then collected and lysed in TRIzol buffer. qPCR was performed. Three independent experiments were performed, and a p value smaller than 0.05 was regarded as statistically significant. (B) The effect of JMJD1C on the CD41 and CD61 expression of K562 cells induced by PMA. Cells were seeded at 2.5 × 105/mL cells and treated with 1 nM PMA for 72 h. Cells were then collected and lysed in TRIzol buffer. qPCR was performed. Three independent experiments were performed, and a p value smaller than 0.05 was regarded as statistically significant. (C) JMJD1C knockdown in MEG-01 cells. Cells were seeded at 2.5 × 105/mL cells and infected with lentivirus carrying JMJD1C shRNA for 72 h. Cells were then collected and lysed in TRIzol buffer. qPCR was performed. Three independent experiments were performed, and a p value smaller than 0.05 was regarded as statistically significant. (D) The effect of JMJD1C on the CD41 and CD61 expression of MEG-01 cells induced by PMA. Cells were seeded at 2.5 × 105/mL cells and treated with 10 nM PMA for 72 h. Cells were then collected and lysed in TRIzol buffer. qPCR was performed. Three independent experiments were performed, and a p value smaller than 0.05 was regarded as statistically significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 5: JMJD1C interacts with Ran. (A,B) Co-immunoprecipitation between JMJD1C and Ran in cell lines K562 (A) and MEG-01 (B). Cells were lysed in P0013 lysis buffer, and immunoprecipitation was performed using the indicated antibodies. The interacting proteins were detected by western blotting.