Fig. 2: PDC-independent DLAT activity is needed for cisplatin-resistant cancer growth.

a Cisplatin response of cancer cells with endogenous DLAT knockdown and rescue expression of human DLAT wildtype (WT) or enzyme-inactive mutant (ΔB; catalytic domain B deleted). DLAT knockdown A549^cisR and KB-3-1^cisR cells expressing DLAT variants were under cisplatin-treated conditions (A549^cisR 2 μg/ml and KB-3-1^cisR 5 μg/ml) for 48 h. Apoptosis and cell viability were assessed by annexin V staining and ATP measurement, respectively. Total DLAT levels in cells with DLAT variants are shown by immunoblotting. b, c DLAT knockdown effect on PDC activity. A549^cisR and KB-3-1^cisR cells with DLAT knockdown were treated with cisplatin for 24 h. PDC activity was determined by monitoring NADH production from PDH reaction (b) and phosphorylation of PDHA1 at S293 (c). d DLAT knockdown effect on PDC assembly. Interaction between E1 (PDH), E3 (DLD), and E2 (DLAT) was determined by E1 co-immunoprecipitation in cells with or without DLAT knockdown and cisplatin treatment. e Effect of PDC inhibition on cisplatin sensitivity. Cells were treated with 5 mM PDK inhibitor dichloroacetate (DCA) and cisplatin for 48 h. Apoptotic cell death and cell viability were measured as described in (a). f Overexpression of ACSS1 in cells lacking DLAT. Acetyl-CoA levels were measured by LC-MS. Apoptotic cell death and cell viability were measured as described in (a). Effect of PDH (g) or DLD (h) knockdown on cisplatin-induced apoptotic cell death and cell viability. Data are mean ± SD from 4 independent biological replicates for apoptosis rates of (a) and cell viability of (g, h left) and 3 independent biological replicates for (b, e, f), cell viability of (a, h right), apoptosis rates of (g, h). P values were determined by one-way ANOVA for all panels. Source data are provided as a Source Data file.