*2.1. Histochrome Does Not A*ff*ect Surface Expression Markers of Human Cardiac Progenitor Cells (hCPCs)*

To evaluate the cytotoxicity of histochrome in human CPCs (hCPCs), hCPCs were treated with different concentrations of histochrome for 24 h. Cell survival was found to be significantly increased for 0.5 μM to 10 μM of histochrome and significantly decreased at concentrations above 100 μM (*p* < 0.01 versus 0 μM; Figure 1B). Based on the data obtained, we determined that histochrome concentration under 50 μM used for the further experiments. No change in the morphology of hCPCs was observed on pretreatment with 0 μM, 5 μM, 10 μM, and 20 μM concentrations of histochrome (Figure 1C). To eliminate the possibility of change in CPC characteristics on pretreatment with histochrome, we investigated typical surface expression markers of hCPCs using fluorescence-activated cell sorting (FACS) analysis. As shown in Figure 1D, histochrome-treated CPCs showed positive expression of cardiac stem cell markers such as mast/stem cell growth factor receptor kit (c-kit), cluster of differentiation 66 (CD166), CD29, CD105, and CD44. However, negative expression was observed for hematopoietic markers, such as CD45 and CD34, in pretreated hCPCs compared to that in control cells.

**Figure 1.** Effects of histochrome treatment on human cardiac progenitor cells (hCPCs) characterization. (**A**) Chemical structure of echinochrome A—active substance of the histochrome drug. (**B**) hCPCs were treated with different concentrations of histochrome for 24 h and viability was measured using cell viability, Proliferation & Cytotoxicity assay (CCK assay). Data are presented as the mean ± standard deviation (SD). \*\*, *p* < 0.01 versus 0 μM, \*\*\*, *p* < 0.001 versus 0 μM. *n* = 6 (**C**) Morphological analysis of hCPCs pretreated with histochrome. Scale bar = 100 μm, (**D**) Expression of stem cell marker by flow cytometric analysis, *n* = 3. Error bars indicate standard effort of the mean (S.E.M)

*2.2. Histochrome Reduced Cellular and Mitochondrial Reactive Oxygen Species (ROS) Levels in hCPCs during H2O2-Induced Oxidative Stress*

To investigate whether pretreating hCPCs with histochrome protects them against oxidative stress, we performed a cellular ROS staining assay. Cellular ROS-tagged green intensity was found to be significantly increased upon exposure to H2O2 (Figure 2A). We observed that pretreatment with histochrome decreased the cellular ROS levels in a dose-dependent manner. The 2',7'–difluorofluorescin diacetate (H2-DFFDA) assay revealed that pretreatment with 10 μM of histochrome significantly decreased cellular ROS levels (Figure 2B). Furthermore, we investigated the effects of pretreatment with histochrome on mitochondrial superoxide production in hCPCs. The increased production of mitochondrial superoxide caused by H2O2 addition was found to be significantly reduced in histochrome-treated hCPCs (Figure 2C). Our data suggested that histochrome has intracellular ROS scavenging activity in hCPCs under oxidative stress.

**Figure 2.** Intracellular reactive oxygen species (ROS) and mitochondrial ROS scavenging activity of histochrome in hCPCs. (**A**) hCPCs were pretreated with histochrome at 0 μM, 5 μM, 10 μM, and 20 μM for 24 h followed by the addition of 600 μM H2O2 for 1 h. Intracellular ROS scavenging activity was measured using CellRox staining. Representative image of increased intensity of CellRox produced by ROS and decreased intensity on pretreating with histochrome. Data are presented as the mean ± SD of three independent experiments. Scale bar = 100 μm, ### *p* < 0.01 versus -H2O2 -histochrome; \* *p* < 0.05; \*\* *p* < 0.01 versus +H2O2 -histochrome, *n* = 3. Error bars indicate S.E.M. (**B**) 2',7'–difluorofluorescin diacetate (H2-DFFDA)assay was used to measure cellular ROS production. \*\*\* *p* < 0.001 versus -H2O2 -histochrome; ###, *p* < 0.001 versus +H2O2 -histochrome, *n* = 3. Error bars indicate S.E.M. (**C**) After pretreatment with histochrome for 24 h, hCPCs were exposed to H2O2 for 1 h and mitochondrial superoxide production was measured with MitoSOX staining. Representative image of the increased intensity of MitoSOX and decreased intensity on pretreatment with histochrome. Scale bar = 100 μm.
