Extended Data Fig. 3: Impact of ROS, proline, and lipids on protein lipoylation.
(a) Immunoblots showing DLAT lipoylation, DLAT, NADK2, and vinculin levels from isogenic ΔNADK2 A549 cells expressing either empty vector or NADK2. Cells were grown in the presence of 0.2 mM proline and treated with the indicated antioxidants for 48 h. GSHee; glutathione ethyl ester (5 mM), NAC; N-acetyl cysteine (1 mM), and Trolox (5 μM). (b) Immunoblotting was done as in (a), but cells were grown in the presence or absence of 0.2 mM proline. (c) Immunoblotting was done as in (a), but cells were grown in the presence of 0.2 mM proline and treated with octanoic acid (50 μM), lipoic acid (50 μM), palmitic acid (100 μM), oleic acid (100 μM), low-density lipoprotein (LDL, 50 μg/ml), or high-density lipoprotein (HDL, 50 μg/ml) for 48 h. (d) Immunoblotting was done as in (a) but in ΔNADK2 A549 cells expressing either empty vector or NADK2. (e) Immunoblotting was done as in (a), but from WT or isogenic ΔNADK2 A549 cells expressing empty vector, NADK2, D161A NADK2, MTS-NADK, or cytosolic NADK. D161A NADK2: kinase-dead NADK2; MTS: Mitochondrial Targeting Sequence. (f) The abundance of intracellular lipoic acid from isogenic ∆NADK2 HeLa or A549 cells expressing either empty vector or NADK2 treated with either vehicle or lipoic acid (50 μM) for 48 h assessed via LC/MS. The peak areas were normalized to the protein amount. (n = 4 biologically independent replicates). Data are presented as the mean ± standard deviation. **P < 0.01 was calculated using a two-tailed Student’s t-test. (g) Immunoblots showing ACP, NADK2, and vinculin levels from WT or ∆NADK2 HeLa cells.
