Fig. 3: DLAT contributes to chemotherapy resistance by controlling mitochondrial ROS. | Nature Communications

Fig. 3: DLAT contributes to chemotherapy resistance by controlling mitochondrial ROS.

From: Non-canonical dihydrolipoyl transacetylase promotes chemotherapy resistance via mitochondrial tetrahydrofolate signaling

Fig. 3

a Effect of targeting DLAT on cellular uptake of cisplatin and cisplatin-mediated DNA damage. Cells were treated with sublethal doses of cisplatin for 24 h, followed by staining with fluorescently labeled antibodies against cisplatin-DNA adducts, phospho-Histone H2A.X, and phospho-53BP1.Effect of DLAT loss and cisplatin treatment on bioenergetics, biosynthesis, and ROS levels. Cells were treated with cisplatin, and ATP, RNA, protein synthesis levels (b) and cellular ROS levels (c) were measured by luminescent assay, metabolic labeling, and DCFDA staining, respectively. d Cisplatin-treated cells with DLAT knockdown were treated with antioxidant NAC (0.5 mM). Cellular ROS (upper), apoptosis (middle), and cell viability (lower) were measured. Effect of mitochondria-targeted antioxidant mito-TEMPO (e) or catalase overexpression (f) on ROS levels, apoptosis, and cell viability in cells with DLAT knockdown and cisplatin. Mito-TEMPO (10 μM) or flag-tagged catalase was introduced in cisplatin-treated cells. Mitochondrial ROS and cytoplasmic hydrogen peroxide were measured by mitoSOX staining and luminescence detection, respectively. g Immunoblotting of apoptosis-associated proteins. Cells with or without DLAT knockdown were treated with sublethal doses of cisplatin for 24 h. h Effect of DLAT loss and NAC treatment on Bcl-xL expression. i Scatter plot of drug sensitivity in KB-3-1cisR and A549cisR representing ROS level, apoptotic rate, or growth inhibition vs -log10(P value). Cells were treated with sublethal doses of drugs (2 μg/ml cisplatin, 50 μM carboplatin, 10 nM gemcitabine, 10 μM etoposide, 0.5 μM pemetrexed, 3 nM paclitaxel, 1 μM erlotinib) for 48 h followed by DCFDA staining (left), annexin V staining (middle), and CellTiter-Glo viability assay (right). Fold changes were obtained by comparing the DLAT knockdown group to the control group. Data are mean ± SD from 3 independent biological replicates for (a–f, i). P values were determined by two-tailed Student’s t-test (a, i) and one-way ANOVA for the rest. Source data are provided as a Source Data file.

Back to article page