*4.4. Pathway Enrichment Analysis*

Integrated Molecular Pathway Level Analysis (IMPaLa, http://impala.molgen.mpg.de/) was used to specify the pathways that are affected by SpD. Only VIP > 1.0 metabolites and Log2(FC) > 1.2 proteins were considered. Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, https://string-db.org) and Cytoscape (downloaded at https://cytoscape.org) were used for clustering molecular networks.

## **5. Conclusions**

The present study investigated the effect of SpD on doxorubicin-treated cardiomyocytes through an integration of metabolic and proteomic analyses. Univariate and multivariate analyses of 1H-NMR spectroscopy data identified the potentially affected metabolites and groups of proteins from SpD-treated cardiomyocytes. Based on the 1H-NMR data, SpD increased glutathione, which regulates intracellular ROS stress. In addition, SpD treatment increased cytosolic and mitochondrial ATP production in cardiomyocytes, which was significantly correlated with increased lactate and decreased acetate levels. Co-treatment with SpD protected doxorubicin-treated cardiomyocytes, reducing the mitochondrial damage of doxorubicin. In contrast, SpD did not inhibit the cytotoxicity of doxorubicin in cancer cells. The integrated metabolomics and proteomics data suggest the involvement of the Akt/mTOR signaling pathway by which SpD might protect cardiomyocytes (Table 3). However, further study is still needed to verify these relationships.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/17/1/2/s1, Figure S1. Spinochrome D (SpD) showed no harmful effect on H9c2 rat cardiac cells. H9c2 rat embryonic cardiomyocytes were treated with 0–200 μM of SpD for 24 h (2 × 104 cells/well, 96-well plates). The cell viability was measured using CCK-8 assay. SpD did not affect on the cell viability of cardiomyocytes. \* mark indicates *p* < 0.05 compared with control. Figure S2. (A) PLS-DA from metabolites of SpD treated AC16 cells. (B) VIP > 0.1 metabolites from SpD treated AC16 cells from PLS-DA. Figure S3. OPLS-DA from metabolites of SpD treated AC16 cells (R2X = 0.333; R2Y = 0.937; Q2 = 0.597). Figure S4. Metabolite sets enrichment overview in SpD treated AC16 cells. Table S1. Genes and metabolites specified for the assigned pathways. Figure S5. STRING analysis revealed that metabolism associated proteins forms clusters around mitochondria (marked as red circles). Figure S6. Spinochrome D (SpD) showed enhanced antioxidant capacity compared with echinochrome A in AC16 and H9c2 cells. (A) SpD and echinochrome A showed no harmful effect on AC16 cells (2 × 104 cells/well, 96-well plate, 24 h). (B) SpD and echinochrome A protected AC16 cells against the cytotoxicity of doxorubicin. (C) SpD showed statistically enhanced antioxidant activity compared with echinochrome A. SpD produced enhanced ATP production in (D) AC16 cells and (E) H9c2 cells compared with echinochrome A. (F) SpD and echinochrome A showed enhanced OCR in AC16 cells. \* mark indicates *p* < 0.05 compared with control. Figure S7. SpD did not inhibit the cytotoxicity of doxorubicin. (A) Mitochondrial membrane potential and mitochondrial calcium was visualized using TMRE and rhod-2 staining in MCF-7 cells. Doxorubicin decreased TMRE intensity and increased rhod-2 intensity in dose-dependent manner. The co-treatment of SpD (10 μM) did not affect doxorubicin-induced tendency of fluorescence intensity. (B) The fluorescence was measured using fluorescence spectrometer. SpD co-treatment did not affect the intensity of TMRE, rhod-2, and DCF-DA in MCF-7 cells. (C) TMRE and rhod-2 fluorescence images from HeLa cells with doxorubicin/SpD. (D) The fluorescence measures indicated that SpD did not affect the intensity of TMRE, rhod-2, and DCF-DA in HeLa cells. Figure S8. SpD inhibited cell migration of cancer cells. 2 × 104 cells of MCF-7 and HeLa were plated in 25-well cell culture plates and scratch wounds were made with 200 μL pipet tips. SpD was treated in 10–200 μM concentrations and incubated for 24 h. Figure S9. Total glutathione and ratio of reduced glutathione to oxidized glutathione (GSH/GSSG). (A) Total glutathione ratio (% of control) from SpD treated AC16 cells. (B) Ratio of reduced glutathione (GSH) to oxidized (GSSG) glutathione normalized by total glutathione concentration from control. The GSH/GSSG assay was measured using GSH/GSSG-GloTM Assay (Promega, WI, USA) according to the product's manual. \* and \*\* mark indicates *p* < 0.05, *p* < 0.01 compared with control, respectively. Supplementary Information 2 lists all proteins detected by mass-spectrometry of SpD treated AC16 cells.

**Author Contributions:** C.S.Y. and H.K.K. conducted the experiments, tests, and data analyses. N.P.M., E.A.V., and S.A.F. isolated and purified the SpD and provided advice for the omics analysis. C.S.Y., H.K.K., V.A.S., and J.H. summarized the work and wrote the manuscript.

**Funding:** This research was funded by the Ministry of Education, Science, and Technology (2010-0020224, 2018R1D1A1A09081767, and 2017K1A3A1A49070056).

**Acknowledgments:** This study was supported by grants from the Priority Research Centers Program, Basic Science Research Program, and International Research & Development Program through the National Research Foundation of Korea (NRF). The study was carried out under support of the Ministry of Education and Science of the Russian Federation (RFMEFI61317X0076) using the equipment of the Collective Facilities Center (The Far Eastern Center for Structural Molecular Research (NMR/MS) PIBOC FEB RAS).

**Conflicts of Interest:** The authors declare no conflict of interest.
