*2.4. Histochrome Protects hCPCs against Oxidative Stress through Downregulation of Pro-Apoptotic Signals and Upregulation of Anti-Apoptotic Signals*

Further, we investigated the expression of apoptosis-related proteins by western blotting. Pretreatment with histochrome was observed to reduce expression of pro-apoptotic protein, Bcl-2 associated X (Bax) while promoting the expression of anti-apoptotic proteins, B-cell lymphoma 2 (Bcl-2), and Bcl-xL. In addition, pretreatment with histochrome remarkably decreased the expression levels of cytochrome C and cleaved-caspase-3 (Figure 4A). Our data suggested that histochrome protects hCPCs against oxidative stress through decreased pro-apoptotic signals and increased anti-apoptotic signals. Further, we checked DNA damage marker, phosphorylated histone (γH2A.X) foci by immunocytochemistry (Figure 4B). We found that pretreatment of hCPCs with histochrome prevented DNA damage caused by oxidative stress. Overall, our results suggested that pretreatment with histochrome regulates cell apoptosis by altering cell survival signals and inhibiting ROS-induced DNA damage.

**Figure 4.** hCPCs pretreated with histochrome show downregulation of pro-apoptotic signals and upregulation of anti-apoptotic signals under the oxidative stress condition. (**A**) hCPCs were pretreated with histochrome for 24 h, oxidative stress was induced in hCPCs by 1 mM H2O2. Expression of apoptosis signaling-related proteins was determined by western blotting. (**B**) Immunofluorescence was performed with the DNA damage marker γH2A.X to quantify DNA damage of hCPCs. Images were captured using a LionHeart FX automated microscope (Biotek, Winooski, VT, USA). \*\*\* *p* < 0.001 versus +H2O2, *n* = 5. Error bars indicate S.E.M.
