Fig 2: Protein level of p-PDHA1 in glioma cells. (A) Western blotting results of p-PDHA1 level in five glioma cell lines. The bar chart indicated the ratio of p-PDHA1 to PDHA1 levels. (B) Cell Counting Kit-8 analysis of TNF-a (50 ng/ml)-induced U251 glioma cells. *P<0.05 vs. Con. (C) Western blot analysis of TNFR1 and p-PDHA1 levels in U251 glioma cells stimulated by TNF-a (50 ng/ml) at different time points. The bar chart indicates the ratio of p-PDHA1 to PDHA1 expression and the expression levels of TNFR1 compared with those of GAPDH. *P<0.05 and #P<0.05 vs. 0 h. p-, phosphorylated; PDHA1, pyruvate dehydrogenase a1; TNF-a, tumor necrosis factor-a; TNFR1, tumor necrosis factor receptor 1; Con, control.

Fig 3: Effects of DCA on the migration and invasion of glioma cells stimulated by TNF-a. (A) Western blot analysis of U251 glioma cells incubated with different concentrations of DCA for 24 h. The bar chart indicates the ratio of p-PDHA1 to PDHA1 expression. *P<0.05 vs. 0 mM. (B) Wound healing assays of NC, DCA, TNF-a and DCA+TNF-a U251 cells. The bar chart is representative of the quantitative results of the wound healing assays. *P<0.05, ^P<0.05 vs. NC; #P<0.05 vs. DCA. (C) Transwell invasion assays of NC, DCA, TNF-a and DCA + TNFa U251 cell groups. The bar chart is indicative of the quantitative results of the Transwell invasion assay. *P<0.05, ^P<0.05 vs. NC; #P<0.05 vs. DCA. (D) Western blot analysis of U251MG cells treated with DCA and/or TNF-a. The bar chart indicated the ratio of p-PDHA1 to PDHA1 expression and the expression levels of TNFR1, MMP2 and MMP14 normalized to those of GAPDH. *P<0.05, ^P<0.05 vs. NC; #P<0.05 vs. DCA. DCA, dichloroacetic acid; TNF-a, tumor necrosis factor-a; p-PDHA1, phosphorylated pyruvate dehydrogenase a1; NC, negative control.

Fig 4: DIC inhibits PDK1 activity in vitro.(A) Molecular docking showing the potential interaction between DIC and PDK1. Left panel, schematic figure showing the surface of the lipoamide-binding pocket, with blue representing a positive charge, red representing a negative charge, and DIC shown in stick form; right panel, the residues from PDK1 that are predicted to interact with DIC and their mode of interactions. (B) In-well kinase assay measuring PDK1 enzymatic activity. Increasing concentrations of DIC (left panel) or DCA (right panel) were added to a 96-well plate. The enzymatic activity of PDK1 was measured by a microplate reader. (C) SKOV3 cells were treated as indicated. The levels of PDK1, PDHA1, and p-PDHA1 were measured using western immunoblotting. ß-Actin was detected as the internal control. Representative western blot images are shown on the left, and the quantitation of PDK1, PDHA1, and p-PDHA1 protein levels relative to ß-actin are shown on the right. (D) A2780 cells were treaed s indicated. The protein levels of PDK1, PDHA1, and p-PDHA1 were examined by western immunoblotting, with ß-actin examined as the internal control. Representative western blot images are shown on the left, and the quantification of protein levels relative to ß-actin are shown on the right. Data are presented as the mean ± SE from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, when compared to nontreated control cells (con).

Fig 5: Knockdown of PDHA1 is associated with the migration and invasion capacities of glioma cells. (A) Western blot analysis of the interference efficiency of U251 cells transfected with siPDHA1. The bar chart indicates the ratio of p-PDHA1 to PDHA1 expression. *P<0.05 vs. siNC. (B) Wound healing assays of U251MG cells treated with si-NC, si-PDHA1-4, TNF-a and siPDHA1 + TNF-a. The migration of glioma cells to the wound surface at 0, 24 and 48 h was observed using the inverted Leica phase contrast microscope (magnification, ×200). The bar chart indicates the quantitative results of the wound healing assays *P<0.05, ^P<0.05 vs. siNC; #P<0.05 vs. TNF-a. (C) Transwell invasion assays of U251 glioma cells in the si-NC, siPDHA1-4, TNF-a and siPDHA1 + TNFa groups. The bar chart indicated the quantitative results of the Transwell invasion assays (magnification, ×100). *P<0.05, ^P<0.05 vs. siNC; #P<0.05 vs. TNF-a. (D) Western blot analysis of siPDHA1-transfected and/or TNF-a-stimulated U251 glioma cells. The bar chart indicates the ratio of p-PDHA1 to PDHA1 expression, as well as the expression levels of TNFR1, MMP2 and MMP14 normalized to those of GAPDH. *P<0.05, ^P<0.05 vs. siNC; #P<0.05 vs. TNF-a + siNC. p-PDHA1, phosphorylated pyruvate dehydrogenase a1; PDHA1, pyruvate dehydrogenase a1; TNF-a, tumor necrosis factor-a; TNFR1, tumor necrosis factor receptor 1.