## 3 Results
### NFAT Reporter Activity of the Chaga Mushroom Extract  
In order to evaluate the immunological activity of the CMW and CME, we tested the nuclear factor of the activated T cell (NFAT) assay. The CTLA-4 Jurkat T cells have a NFAT response when CTLA-4/CD80 is blocked by anti-CTLA-4 antibodies or small molecules sent to signal expression of luciferase. Once luciferase is expressed, it produces light in an enzymatic assay, and the amount of light can be measured. CMW and CME were treated to a four-fold dilution (1.56 \(\upmu\)g/mL to 12.5 \(\upmu\)g/mL) on a co-culture well plate with CTLA-4 Jurkat T cells: CD80/aAPC CHO-K1 cells (1:1). We observed that the CMW and CME blocked CTLA-4/CD80 interaction. The luminescence of the CTLA-4/CD80 reporter assays that were treated with CMW and CME significantly increased (\(\sim\)3 fold and \(\sim\)4.5 fold, respectively) at 12.5 \(\upmu\)g/mL, higher than the negative control \"cell only\", with EC\({}_{50}\) values of 3.12 \(\upmu\)g/mL and 3.95 \(\upmu\)g/mL, respectively. These results supported our observation of the ELISA assay results (Figure 2).  
Figure 1: Blocking effect of the CTLA-4/CD80 interaction of CMW and CME by competitive enzyme-linked immunosorbent assay (ELISA). Effect of anti-CTLA-4 neutralizing antibody (**A**); water extract (**B**); and 70% ethanol extract (**C**) of Chaga mushroom on the CTLA-4/CD80 binding activity. The relative binding activity was normalized to the relative percentage of the vehicle control group. The half-maximal inhibitory concentration (IC\({}_{50}\)) was calculated using the Prism “log[inhibitor] vs. the normalized response” equation. All results are presented as the mean of three independent biological replicates, with \({}^{\star}\)\(p\)\(<\) 0.05, \({}^{\star\star}\)\(p\)\(<\) 0.01, \({}^{\star\star\star}\)\(p\)\(<\) 0.001, compared with the vehicle control group.  
Evaluation of Cytotoxicityicity of the Chaga Mushroom Extract in CTLA-4 Jurkat T and CD80/aAPC CHO-K1 Cells  
To identify how CMW and CME can affect the cell viability of both CTLA-4 Jurkat T cells and CD80/aAPC CHO-K1 cells, they were used in the cell-based assay for CTLA-4/CD80 inhibitor screening. Several concentrations of CMW and CME were treated on CTLA-4 Jurkat T and CD80/aAPC CHO-K1 cells for 24 h. Then, the cell viability was assessed using a Cell Counting Kit-8 (CCK) assay. In all tested concentrations, neither the CMW nor the CME showed an effect on the cell viability of CTLA-4 Jurkat T cells or CD80/aAPC CHO-K1 cells. These results showed no cytotoxicity at the concentrations used in the previously described NFAT reporter assay (Figure 3).  
Figure 3: Cell viability effects of the Chaga mushroom/water extract (CMW) and Chaga mushroom/70% EtOH extract (CME) on CTLA-4 Jurkat T and CD80/aAPC CHO-K1 cells. CME (**A**) and CMW (**B**) were treated with various concentrations (6.25 to 200 ug/mL) on CTLA-4 Jurkat T and CD80/aAPC CHO-K1 cells for 24 h, and the cell viability was assessed using a CCK8 assay. All results are presented as the mean of three independent biological replicates, with \({}^{*}p<0.05\), \({}^{**}p<0.01\), compared with the vehicle control group.  
Figure 2: Nuclear factor of the activated T cell (NFAT) luciferase reporter activity by CTLA-4/NFAT Reporter- Jurkat T cells treated with Chaga mushroom/water extract (CMW) and Chaga mushroom/70% EtOH extract (CME). Fold luminescence of Jurkat T cells treated with CMW (**A**) and CME (**B**) at several concentrations (1.56 to 12.5 ug/mL). The half-maximal effective concentration (EC\({}_{50}\)) was calculated using the Prism “log[agonist] vs. normalized response” equation. All results are presented as the mean of three independent biological replicates and compared with the vehicle control group.