# Immunomodulatory Effects of Pentoxifylline: Profiling Data Based on RAW 264.7 Cellular Signaling
## 3 Results

Table 1: All 132 antibodies used in this study. (90.9%), and cyclin D2 (88.7%) was expressed than untreated controls. Changes of PCNA, PLK4, p14, and p16 were measured within \(\pm\)5% in response to PTX, which were analogous with control housekeeping proteins (Figure 1a). PTX inhibits the proliferation of RAW 264.7 cells. **Expression of cMyc/MAX/MAD signaling proteins**. Slightly increased expression of cMyc (106.4%) and MAX (108.8%) is expressed in samples treated for 24 h. The expression level of MAD (105.7%) also increased slightly after treatment for 12 h. PTX slightly enhanced cMyc/MAX signaling and participated in transcriptional control in RAW 264.7 cells (Figure 1b). **Expression of p53/Rb/E2F signaling proteins**. Expression of p53 (86.4%), Rb-1 (91.7%), E2F-1 (88.3%), MDM2 (85.1%), CDK4 (92.3%), and p21 (90.2%) is shown. PTX inhibits p53/Rb/E2F signaling leading to cellular proliferation (Figure 1c). Figure 1: (**a**) Changes of proliferation-related proteins after PTX treatment in RAW 264.7 cells; (**b**) changes to cMyc/MAX/MAD signaling proteins after PTX treatment in RAW 264.7 cells; (**c**) changes to p53/Rb/E2F signaling-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. **Expression of epigenetic modification-related proteins. ** Slightly increased expression of histone H1 (108.6%) and MBD4 (111.0%) but no significant change in the expression of KDM4D (95.1-103.6%) is found. A decrease in the expression of DMAP1 (88.0%), DNMT1 (94.5%), and HDAC-10 (94.9%) is also found. PTX may reduce DNA methylation and activate DNA transcription in RAW 264.7 cells. Therefore, PTX can be associated with epigenetic modifications and the regulation of gene expression. MBD4 may function to mediate the biological consequences of the methylation signal. MBD4 is similar in protein sequence to bacterial DNA repair enzymes and has been shown to perform some functions in DNA repair (Figure 2a). **Expression of translation-related proteins. ** Slightly reduced expression of eIF5A1 (92.5%) is found. The expression levels of DHS (95%) and DOHH (95.2%) are similar to  
Figure 2: (**a**) Changes to epigenetic modification-related proteins after PTX treatment in RAW 264.7 cells; (**b**) changes to protein translation-related proteins after PTX treatment in RAW 264.7 cells; (**c**) changes in cellular differentiation-related proteins after PTX treatment in RAW 264.7 cells. Left-sided graph shows changes in protein expression 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 916 kb). control proteins. PTX may reduce protein translation for cellular proliferation and other functions (Figure 2b). **Expression of cellular differentiation-related proteins. ** Slightly reduced expression levels of the differentiation-related proteins including PLC-82 (88.1%), TGase-2 (94.6%), HXK II (92.4%), Jagged-2 (93.8%), and GLI-1 (94.5%) is found. The expression of Notch-1 did not show significant changes, remaining similar to those of the housekeeping proteins. PTX treatment down-regulates cellular differentiation in RAW264.7 cells (Figure 2c). **Expression of RAS signaling proteins. ** PTX slightly reduced the expressions levels of V-Ki-ras2 KRAS (92.0%), NRAS (94.2%), and GTPase HRas (HRAS, 94.3%). MEKK (107.6%) expression was increased in the 12-hour-treated group but decreased with time similar to that of housekeeping proteins. The expression of ERK-1 (108.9%) increased in the 24-hour-treated group.