Table 1.
Assignment of metabolites observed by 1H-NMR in cancer cell lysates.
Fig 1.
Analysis of choline kinase-α (CK-α), estrogen receptor- and LC3 in CK-α-knockdown cells in MCF-7 and MCF-7/TAM.
(A) Flow cytometric analysis of GFP positive cells in MCF-7/shCK-α and MCF-7/TAM/shCK-α transduced with lentivirus containing CK-α shRNA and GFP. (B) Images of GFP expression in MCF-7/shCK-α and MCF-7/TAM/shCK-α. (C) RT-PCR analysis of CK-α and CK-β in MCF-7, MCF-7/TAM, MCF-7/shCK-α and MCF-7/TAM/shCK-α. (D) Western blot analysis of CK-α in MCF-7, MCF-7/TAM, MCF-7/shCK-α and MCF-7/TAM/shCK-α. (E) Western blot analysis of ER-α in MCF-7, MCF-7/TAM, MCF-7/shCK-α and MCF-7/TAM/shCK-α. (F) Western blot analysis of LC3I/II in MCF-7, MCF-7/TAM, MCF-7/shCK-α and MCF-7/TAM/shCK-α. Data are presented as the mean ± standard error. *p<0.05, 0.001<**p<0.05, ***p<0.001.
Fig 2.
PCA and PLS-DA for MCF-7, MCF-7/shCK-α, MCF-7/TAM and MCF-7/TAM/shCK-α groups.
(A) PCA for the inspection and overview of the data set. (B) Score plot for the investigation of group differentiation among MCF-7 (MC, n = 4, green), MCF-7/shCK-α (Msh, n = 6, blue), MCF-7/TAM (MT, n = 4, red), and MCF-7/TMA/shCK-α (MTsh, n = 5, yellow) cell groups. (C) VIP values as a measure of the discriminatory potential of the individual metabolites in the group differentiation. (UA: unknown resonance A, lactate, UE: unknown resonance E, glutamine, formate, AXP: AMP/ADP/ATP, UXP: UMP/UDP/UTP, MAU: maleate/AXP/UXP, UC: unknown resonance C, NAG: N-acetyl-aspartate/-amino acid/glutamate, Glx: glutamate/glutamine, LIV: leucine/isoleucine/valine). (D) Loading plot showing the relative contribution of the metabolites to the scores in the score plot. By matching the positions in the score plot and loading plot, the correlations between the individual metabolites and the cell groups were determined.
Fig 3.
Comparison of the concentrations of the metabolites with VIP>1 among the MCF-7, MCF-7/shCK-α, MCF-7/TAM, and MCF-7/TAM/shCK-α cell groups.
Increased lactate, glycine, phosphocholine, glutamine, and succinate in MCF-7/TAM relative to the other cell groups. Increased myo-inositol in MCF-7/shCK-α relative to the other cells. Non-detectable formate in MCF-7/TAM and MCF-7/TAM/shCK-α. Non-detectable fumarate in 7/shCK-α and MCF-7/TAM and MCF-7/TAM/shCK-α. Decreased AXP in MCF-7/shCK-α cells and MCF-7/TAM/shCK-α relative to MCF-7 and MCF-7/TAM. The spectra were normalized against total intensity and averaged over the samples in MCF-7, MCF-7/shCK-α, MCF-7/TAM and MCF-7/TAM/shCK-α. All values are presented as the mean ± standard error. *p<0.05, ** 0.001<p<0.05, *** p<0.001.