*2.1. Validation and Characterization of PADI2 Knockout U937 Cells*

We confirmed the protein expression of PADI2 was dramatically decreased after gene knockout using the CRISPR–Cas system (Figure 1A). PADI2 and PADI4 are homologous in their structures and amino acid sequences in human [2]. We found that the protein expression of PADI4 did not change in the U937 cells after PADI2 knockout compared with those in the controls (Figure 1A). Next, we analyzed the protein levels for cit-H3. In the U397 cells, the differentiated macrophages, and the macrophages stimulated with LPS, the protein levels of cit-H3 were not different between the PADI2 knockout group and the controls (Figure 1B). Sun et al. reported that PADI4 could citrullinate nuclear factor κB (NF-κB) p65 and enhance its nuclear translocation and transcriptional activity [11]. We found that the protein levels of the citrullinated p65 were decreased in the nuclear extract of macrophages stimulated with LPS in the PADI2 knockout group compared with in the controls (Figure 1C,D).

**Figure 1.** Validation of peptidylarginine deiminase 2 (PADI2) knockout U937 cells and their effects on histone H3 and nuclear factor kappa B (NF-κB) p65 citrullination. (**A**) Comparison of the relative protein expression levels of PADI2 and PADI4 with those of the control. The U937 cells were transfected with CRISPR–Cas9 plasmids containing gRNA that targets PADI2 or control CRISPR-Cas9 plasmids as

the control group. The protein expression levels of PADI2 decreased dramatically, but those of PADI4 did not changed after PADI2 gene knockout (PADI2 knockout group). (**B**) Comparison of the citrullinated histone H3 (cit-H3)/actin ratios of the U937 cells, the differentiated macrophages, and the macrophages stimulated with 20 ng/mL lipopolysaccharides (LPS). The protein level of cit-H3 did not change in the U937 cells, the differentiated macrophages, and the macrophages stimulated with 20 ng/mL LPS for 24 h in the PADI2 knockout group. (**C**) Comparison of the citrullinated p65 subunit of NF-κB (cit-p65) ratios of the LPS-stimulated macrophages. The citrullinated protein was obtained from the nuclear extract of LPS-stimulated macrophages by immunoprecipitation using ACPAs-conjugated protein G Sepharose beads. The protein expression of the cit-p65 in the immunoprecipitates was then analyzed by Western blotting using anti-p65 antibodies as a probe. (**D**) Representative images showing the relative protein expression levels of PADI2 and PADI4; citrullinated histone H3 of the U937 cells, the differentiated macrophages, and the macrophages stimulated with LPS and cit-p65 in the immunoprecipitates of the LPS-stimulated macrophages in the PADI2 knockout group and the control group.
