## 4 Conclusions  
This study presented structural and biochemical changes in cordycepin-induced HeLa cell apoptosis and investigated its apoptosis-inducing mechanism. DIC and fluorescence microscopic imaging was used to demonstrate the effectiveness of cordycepin in inducing HeLa cell apoptosis, and to illustrate the morphological changes of cells undergoing apoptosis. SERS was used to reveal the detailed biochemical changes, such as nucleic acid, proteins, lipids, RNA, etc. Specifically, SERS characteristic peaks at 805 and 1438 cm\({}^{-1}\), which were assigned to RNA, continued to decrease significantly from normal to the early apoptosis stage, and then to the late apoptosis stage. Therefore, cordycepin induced HeLa cell apoptosis through interfering with RNA production, potentially by restraining the translation of RNA encoding ribosomal proteins. Moreover, apoptotic cells and their apoptosis stage could be easily differentiated by PCA. The natural ingredient cordycepin was a promising candidate in the treatment of cervical cancer. Our biological model of cell apoptosis in vitro also provided a reference for clinical treatment of cancer and anticancer drug development. In addition, the non-invasive, rapid SERS technique, combined with PCA analysis, was a powerful tool in the investigation of cell apoptosis mechanisms and anticancer drug screening. The following are available online at [[http://www.mdpi.com/2076-3417/9/19/3990/s1](http://www.mdpi.com/2076-3417/9/19/3990/s1)]([http://www.mdpi.com/2076-3417/9/19/3990/s1](http://www.mdpi.com/2076-3417/9/19/3990/s1)), Figure S1: The TEM images of silver nanoparticles, Figure S2: The particle size distribution of the silver colloid, Figure S3: The SERS spectrum of cordycepin, Figure S4: DIC and fluorescence microscopic images of HeLa cells in group 1-3. The DIC images of cells co-cultured with: (a) 20 \(\mathrm{\SIUnitSymbolMicro g}\)/ml silver nanoparticles, (d) 60 \(\mathrm{\SIUnitSymbolMicro g}\)/ml silver nanoparticles, (g) 200 \(\mathrm{\SIUnitSymbolMicro g}\)/ml silver nanoparticles; the fluorescence images of cells co-cultured with: (b) 20 \(\mathrm{\SIUnitSymbolMicro g}\)/ml silver nanoparticles, (e) 60 \(\mathrm{\SIUnitSymbolMicro g}\)/ml silver nanoparticles, (h) 200 \(\mathrm{\SIUnitSymbolMicro g}\)/ml silver nanoparticles; the Imgered DIC and fluorescence images were shown in (c), (f) and (i) respectively. In the DIC images, the bright or dark dots appeared were silver nanoparticles that resided on the bottom of the petri dish or those that had been endocytosed into the cells, Figure S5: DIC and fluorescence microscopic images of HeLa cells in group 4-6. The DIC images of cells co-cultured with: (a) 20 \(\mathrm{\SIUnitSymbolMicro g}\)/ml silver nanoparticles, (d) 60 \(\mathrm{\SIUnitSymbolMicro g}\)/ml silver nanoparticles, (g) 200 \(\mathrm{\SIUnitSymbolMicro g}\)/ml silver nanoparticles; the fluorescence images of cells co-cultured with: (b) 20 \(\mathrm{\SIUnitSymbolMicro g}\)/ml silver nanoparticles, (e) 60 \(\mathrm{\SIUnitSymbolMicro g}\)/ml silver nanoparticles, (h) 200 \(\mathrm{\SIUnitSymbolMicro g}\)/ml silver nanoparticles; the Imgered DIC and fluorescence images were shown in (c), (f) and (i) respectively. In the DIC images, the bright or dark dots appeared were silver nanoparticles that resided on the bottom of the petri dish or those that had been endocytosed into the cells, Figure S6: SERS spectral changes of HeLa cells treated with cordycepin in the early and late stage of apoptosis. (a): The average SERS spectrum of the control group; (b): the average SERS spectrum of the cordycepin-12h group; (c): the average SERS spectrum of the cordycepin-48h group; (d): spectral difference between (b) and (a); (e): spectral difference between (c) and (a). The x-axis was Raman shift and y-axis was the intensity in arbitrary unit (a.u.). Table S1: A summary of treatment and results of the Ag-cell apoptosis experiment. Concept formation P.M., L.X., H.C. and J.L. ; experimental design P.M. and L.X. ; data acquisition L.X., and P.M.; data analysis L.X.