*2.2. E*ff*ects of PADI2 Knockout on Proinflammatory Cytokines Expression and Secretion*

In Figure 2A, we found that the gene expression levels of *IL-1*β, *IL-6*, and *TNF-*α were increased in the differentiated macrophages compared with in the U937 cells in both the PADI2 knockout group and the control group. The addition of LPS further increased the mRNA expression levels of *IL-1*β (23.9 ± 0.2 vs. 23.3 ± 0.3; *p* = 0.017) and *IL-6* (17.2 ± 0.2 vs. 15.5 ± 0.1; *p* < 0.001), but not those of *TNF-*α (19.1 ± 0.5 vs. 19.6 ± 0.3; *p* = 0.135) in the control group. The addition of LPS did not affected the mRNA expression levels of f *IL-1*β, *IL-6*, and *TNF-*α in the PADI2 knockout group.

In the U937 cells, the PADI2 knockout decreased the gene expression levels of *IL-1*β and *TNF-*α compared with those in the controls. In the differentiated macrophages and the macrophages stimulated with LPS, the PADI2 knockout decreased the gene expression levels of *IL-1*β and *IL-6* compared with those in the controls.

In the U937 cells, the secretion levels of IL-1β, IL-6, and TNF-α were very low in both the PADI2 knockout group and the control group (Figure 2B). In both groups, the differentiated macrophages secreted the increased levels of IL-1β, IL-6, and TNF-α compared with the U937 cells. The addition of LPS further increased the secretion levels of IL-1β, IL-6, and TNF-α in both groups.

In the differentiated macrophages and the macrophages stimulated with LPS, PADI2 knockout significantly decreased the cytokine secretion levels of IL-1β, IL-6, and TNF-α compared with in the controls.

#### *2.3. E*ff*ects of PADI2 Knockout on Cell Apoptosis and Adhesion*

As expected, the apoptotic rate was significantly elevated in the differentiated macrophages compared with in the U937 cells. The addition of LPS further increased the apoptotic rate of the macrophages in both the PADI2 knockout group and the control group (Figure 3A,B). The PADI2 knockout group significantly decreased the apoptotic rates in the U937 cells, the differentiated macrophages, and the macrophages stimulated with LPS compared with the control group. We also noticed that the PADI2 knockout macrophages were more easily detached during trypsinization compared with the controls. Stefanelli et al. showed that protein citrullination could alter focal adhesion stability [12]. Therefore, we speculated that PADI2 knockout might also impair the macrophage adhesion. Using a commercially available fibrinogen-coated plate, we found that the PADI2 knockout group had impaired cell adhesion ability compared with the control group (Figure 3C).

**Figure 2.** Effects of PADI2 knockout on inflammatory cytokines expression and secretion. (**A**) The mRNA expression levels of *IL-1*β, *IL-6*, and tumor necrosis factor-alpha (*TNF-*α) in the U937 cells, the differentiated macrophages, and the macrophages stimulated with LPS in the PADI2 knockout group compared with those in the controls. (**B**) The cytokines secretion levels of IL-1β, IL-6, and TNF-α in the U937 cells, the differentiated macrophages, and the macrophages stimulated with LPS transfected in the PADI2 knockout group compared with those in the control group. ✩ *p* < 0.05 compared with the U937 cells; \* *p* < 0.05 compared with the differentiated macrophages.

**Figure 3.** Effects of PADI2 knockout on macrophages apoptosis and adhesion. (**A**) The apoptosis rates of the U937 cells, the differentiated macrophages, and the macrophages stimulated with LPS for 24 h

in the PADI2 knockout and control groups. The apoptotic rates of these cells were measured using flow cytometry analysis. The cells apoptosis was defined as % of annexin V staining. (**B**) Comparison of cell apoptosis in the macrophages stimulated with LPS in the PADI2 knockout group and the control group. It was shown than cell apoptosis in the macrophages stimulated with LPS in the PADI2 knockout group was decreased compared with in the control group. (**C**) The adhesion ability in the differentiated macrophages from the PADI2 knockout group and the control group using a fibrinogen-coated plate. ✩ *p* < 0.05 compared with the U937 cells; \* *p* < 0.05 compared with the differentiated macrophages.
