# Immunomodulatory Effects of Pentoxifylline: Profiling Data Based on RAW 264.7 Cellular Signaling
## 3 Results
The expression level of mTOR (94.0%) was decreased in the 12-hour-treated sample, while that of pAKT1/2/3 (89.1%) markedly decreased with time. Changes in JNK-1, Rab, and PKC expression were similar to housekeeping proteins. RAS signaling appeared to be reduced by PTX (Figure 3a). **Figure 3. (a). Changes to RAS signaling-related proteins after PTX treatment in RAW 264.7 cells; (b) changes to NFxB signaling-related proteins after PTX treatment in RAW 264.7 cells; (c) changes to growth factor-related proteins after PTX treatment in RAW 264.7 cells. Left-sided graph shows changes of protein expressions according to the application time of PTX, blue bar; control group, red bar; 12 h treatment of PTX, green bar; 24 h treatment of PTX, purple bar; 48 h treatment of PTX. Right-sided polygonal graph shows protein expression profiles after 12 h treatment of PTX. (JPG 1.05 MB). **  
**Expression of NFkB signaling proteins**. A decrease in the expression of NFkB signaling proteins is shown. After PTX treatment, TNF\(\alpha\), GADD 153 decreased to a minimum of \(\pm\)5%, but the expression levels of NFkB (84.9%), IKK (94.2%), PAKT (89.1%), PGC-1\(\alpha\) (86.2%), and p38 (84.4%) were significantly reduced. RAW 264.7 cells treated with PTX showed a slight decrease in the expression of MDR (92.4%) and a slight rise in the expression of ERK1 (108.9%). The expression levels of GADD45 were slightly increased (105.0%) in the 12-hour-treated sample; however, levels decreased with time. NFkB is a key regulator of immune responses and inflammation. The NFkB signaling system is responsive to a number of stimuli. In our IP-HPLC result, protein expression related to NFkB signaling did not increase significantly. These results indicated that NFkB signaling was affected by PTX treatment and that the cells were in a stress-free state (Figure 3b). **Expression of growth factor-related proteins**. Slight increases in the expression of IGF1 (107.2%), HER1 (107.5%), and HER2 (107.3%) were observed. Conversely, the expression levels of FGF1 (88.8%), FGF2 (91.9%), and ER\(\beta\) (92.8%) decreased in RAW 264.7 cells treated with PTX. The expression of TGF-81 (96.2-104.4%), CTGF (95.2-105.1%), and Met (95.9-104.6%) changed only minimally to less than \(\pm\)5% (Figure 3c). **Expression of inflammation-related proteins**. A decrease in IL-6 (92.4%) and IL-1 (88.9%). IL-28 (97.7-105.9%) showed no significant change. The expression level of IL-10 significantly increased to 123.5% by PTX treatment. MMPs expression levels after PTX treatment varied; MMP-1, MMP-2, MMP-3, and MMP-9 were increased to 108.4%, 109.1%, 117.0%, and 117%, respectively, while MMP-10 revealed a minimal change of less than \(\pm\)5%, and MMP-12 decreased in the 12-hour-treated sample to 93.9%. The expression levels of LTA4H, CXCR4, M-CSF, MCP-1, hepcidin, and cathepsin K were dramatically reduced by PTX to 91.2%, 82.3%, 87.7%, 93.8%, 94.2%, and 92.7%, respectively, but TNF\(\alpha\) and TLR3 did not show significant changes. COX-2 (91.0%) expression was reduced by PTX treatment; however, that of CRP-1 was slightly increased in the 12-hour-treated sample (109.9%), which decreased to 101.4% with time. These results suggest that PTX downregulates LTA4H, CXCR4, ILs, and MMPs and induces anti-inflammatory signaling in RAW 264.7 cells without affecting TNF\(\alpha\) (Figure 4a,b). **Expression of immunity-related proteins**. Significant increases in the expression of cluster of differentiation 4 (CD4, 112.1%) and CD20 (106.4%) were observed. The expression levels of CD31 and CD68 showed minimal changes of less than \(\pm\)5%, which was analogous to that of the housekeeping proteins. The expression levels of CD3 and CD28 after PTX treatment were 87.4% and 91.6%, respectively. PTX is a phosphodiesterase inhibitor, the exact mechanism of various immunomodulatory functions has not been elucidated. Importantly, the expression levels of CD3 and CD28 were reduced by PTX, however, the exact mechanism could not be found.