## 3 Results and Discussion
### PCA and Histogram Analysis  
PCA was further used to discriminate the two sets of spectra in terms of statistics. PCA is a data analysis technique used to discriminate against a multivariate data set. In this study, PCA was used to analyze the scatter of the datasets acquired from the two different groups of the experiment. The PCA score plot (Figure 4A) demonstrates that the second derivative spectra of mycoplasma-decontaminated cells and -contaminated cells could be separated. The discrimination is mainly accounted for by PC1 (66%, \(x\)-axis), and lesser separation along PC2 (22%, \(y\)-axis). Analysis of the PCA loading plots (Figure 4B,C) was used to determine the regions of the FTIR spectrum, which mostly contributed to the clustering (Figure 4A). The secondary spectra of  
Figure 4: **(A)** Principal component analysis (PCA) score plot on PC1 and PC2 and **(B)** their relevant loading plots from the lipid and **(C)** protein regions represent the wavenumbers discriminating the differences between decontaminated (67 spectra from triplicate samples) and contaminated (58 spectra from triplicate samples) HepG2 cells. The wavenumbers with loading scores \(\geq 0.1\) or \(\leq-0.1\) are significant. the contaminated cells could be distinguished from the decontaminated cells by the positive PC1 scores (Figure 4A). The positive values of PC1 loading in the lipid region (Figure 4B) at 2969 cm\({}^{-1}\) (assigned to \(\upgamma_{\rm as}\) of H-CO), 2941 cm\({}^{-1}\) (\(\upgamma_{\rm as}\) of CH\({}_{3}\)[27]), 2908 cm\({}^{-1}\) (assigned to \(\upgamma_{\rm s}\) of CH\({}_{2}\) and CH\({}_{3}\) of lipid [28]), 2865 cm\({}^{-1}\) (C\(-\)H stretching [29]), and 2838 cm\({}^{-1}\) (\(\upgamma_{\rm s}\) of CH\({}_{3}\)[27]) were heavily loaded for PC1, which separated the negative score of the spectra of the decontaminated cells from the positive score of the spectra of the contaminated cells (Figure 4B). The high negative of PC1 loading at 2923 and 2852 cm\({}^{-1}\) (assigned as the C\(-\)H stretching of lipids band) could be due to a higher lipid in mycoplasma-contaminated HepG2 cells. Moreover, the other negative values of PC1 loading of the respective peaks at 3005 and 2956 cm\({}^{-1}\) (assigned as unsaturated lipid [21; 22] and saturated fatty acid [22]) were loaded for PC1, which separated the positive score of the spectra of the contaminated cells from the negative score of the spectra of the decontaminated cells. The high positive values of PC1 loading in the protein region (Figure 4C) at 1648 cm\({}^{-1}\) (assigned as random coil [30]), 1544 cm\({}^{-1}\) (\(\upalpha\)-helix of amide II band [31]), and 1639 cm\({}^{-1}\) (\(\upbeta\)-sheet structure of amide I band of proteins [30; 32]), respectively, were heavily loaded for PC1, which separate the negative score of the spectra of the decontaminated cells from the positive score of the spectra of the contaminated cells (Figure 4C). The other positive values of PC1 loading were found at 1720 cm\({}^{-1}\) (assigned as C\(=\)O stretching band of (i) fatty acid ester or (ii) nucleic acid [29]), 1624 cm\({}^{-1}\) (\(\upbeta\)-sheet of amide I band [30]), 1512 cm\({}^{-1}\) (8 CH (C\(-\)H in phenyl ring) [29]) and 1477 cm\({}^{-1}\) (CH\({}_{2}\) bending of the methylene chains in lipids; orthorhombic stage or packed stage of the lipid bilayer [24; 29]). These data indicated that the \(\upalpha\)-helix and \(\upbeta\)-sheet bands of protein structures were most strongly responsible for discriminating the decontaminated cells from the contaminated cells. Our result showed that all of the discriminating wavenumbers are from both lipid and protein regions (Figure 4B,C, and Table 2). No discriminating wavenumber from the nucleic acid region was observed. The result is in agreement with data from the inspection of second derivative spectra, suggesting that differences between mycoplasma-decontaminated and -contaminated HepG2 cells are affected by the intracellular lipid and protein contents.