Journal: The Journal of Biological Chemistry

Article Title: Cardiolipin-induced activation of pyruvate dehydrogenase links mitochondrial lipid biosynthesis to TCA cycle function

doi: 10.1074/jbc.RA119.009037

Figure Lengend Snippet: Decreased activity of PDH in tafazzin-deficient cells. A, PDH activity was assayed in isolated mitochondria as described under “Experimental procedures.” Data shown are mean ± S.D. (n = 3). *, p < 0.05. B, levels of PDH-E1α in mitochondrial extracts were determined by Western blot analysis with anti-PDH-E1α antibody. 50 μg of total protein from each sample was loaded onto an SDS gel under reducing conditions, and the mitochondrial protein Ndufb6 was used as a loading control. C, PDH activity was assayed in heart tissue extracted from WT and TAZ-KO mice. Each column represents a separate mouse. Data shown are mean ± S.D. (n = 2). D, WT and TAZ-KO cells were seeded in DMEM (without glucose, pyruvate, or glutamine/1% FCS), followed by 3-h incubation in Dulbecco's PBS (Life Technologies) supplemented with 100 μm [1-14C]pyruvate, and the PDH flux assay was performed in the presence or absence of 5 mm DCA. PDH activity was assayed by measuring the release of 14CO2 from [1-14C]pyruvate as described under “Experimental procedures.” Data shown are mean ± S.D. (n = 3). *, p < 0.05.

Article Snippet: PDH bound to lipids on the membrane was detected by immunoblotting using primary antibody (Assaypro) against the PDH complex (1:1000) and secondary antibody conjugated to horseradish peroxidase (1:3000).

Techniques: Activity Assay, Isolation, Western Blot, SDS-Gel, Incubation, Flux Assay

Journal: The Journal of Biological Chemistry

Article Title: Cardiolipin-induced activation of pyruvate dehydrogenase links mitochondrial lipid biosynthesis to TCA cycle function

doi: 10.1074/jbc.RA119.009037

Figure Lengend Snippet: CL binds to PDH and increases its activity. A, digitonin-solubilized mitochondria from WT and TAZ-KO cells were treated with or without CL. PDH activity was assayed as described under “Experimental procedures.” B, purified PDH complex (Sigma) was incubated with exogenous CL or phosphatidic acid (PA). PDH activity was assayed as described under “Experimental procedures”. Data shown are mean ± S.D. (n = 3). *, p < 0.05. C, the indicated lipids CL, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 3,4,5-trisphosphate (PIP3), and phosphatidic acid were serially diluted and spotted onto a nitrocellulose membrane, which was incubated overnight in buffer containing 10 μg of PDH complex. Interactions were detected by immunoblotting with an antibody against the PDH complex.

Article Snippet: PDH bound to lipids on the membrane was detected by immunoblotting using primary antibody (Assaypro) against the PDH complex (1:1000) and secondary antibody conjugated to horseradish peroxidase (1:3000).

Techniques: Activity Assay, Purification, Incubation, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Effect of Huaier on the proliferation and apoptosis of human gastric cancer cells through modulation of the PI3K/AKT signaling pathway

doi: 10.3892/etm.2015.2600

Figure Lengend Snippet: Effect of Huaier extract on the protein expression of AKT1, PI3K, PTEN, PDK1, caspase-9 and Bcl-2 in SGC7901 cells. The expression of GAPDH was used as an internal control. The SGC7901 cells were treated with 0, 2.5 and 5 mg/ml Huaier for 24 h, and (A) western blotting was used to analyze the protein expression. (B) Quantification of the data. The experiment was performed in triplicate, and the data are expressed as the mean ± standard deviation of the three separate experiments. *P<0.05 and **P<0.01, compared with the control. p-, phosphorylated-; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase; PDK1, pyruvate dehydrogenase kinase isoform 1; Bcl-2, B-cell lymphoma 2; BAD, Bcl-2-associated death promoter.

Article Snippet: Primary monoclonal antibodies against AKT (1:1,000; 10176-2-AP), PDK1 (1:1,000; 10026-1-AP), Bcl-2-associated death promoter (BAD; 1:1,000; 10435-1-AP) and GAPDH (1:4,000; 60004-1-Ig) were purchased from Proteintech (Manchester, UK), while monoclonal antibodies against p-AKT (The-308; 1:800; 2118-1), p-AKT (Ser-473; 1:800; 2214-1), PI3K (1:1,000; 1593-S), PTEN (1:1,000; 5171-1), p-PTEN (1:1,000; 2134-1), Bcl-2 (1:1,000; 10026-1-AP), pro-caspase-9 (1:1,000; 1023-1) and cyclin B1 (1:2,000; 1495-1) were obtained from Abcam (Cambridge, UK), and monoclonal anti-cleaved-caspase-9 (1:1,000; sc-22182) was from Santa Cruz Biotechnology, Inc. (Shanghai, China).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Effect of Huaier on the proliferation and apoptosis of human gastric cancer cells through modulation of the PI3K/AKT signaling pathway

doi: 10.3892/etm.2015.2600

Figure Lengend Snippet: Effect of Huaier extract on the protein expression of AKT1, PI3K, PTEN, PDK1, caspase-9 and Bcl-2. The expression of GAPDH was used as an internal control. MKN45 cells were treated with 0, 2.5 and 5 mg/ml for 24 h, and (A) western blotting was used to analyze the protein expression. (B) Quantification of the data. The experiment was performed in triplicate, and the data are expressed as the mean ± standard deviation of the three separate experiments. *P<0.05 and **P<0.01, compared with the control. p-, phosphorylated-; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase; PDK1, pyruvate dehydrogenase kinase isoform 1; Bcl-2, B-cell lymphoma 2; BAD, Bcl-2-associated death promoter.

Article Snippet: Primary monoclonal antibodies against AKT (1:1,000; 10176-2-AP), PDK1 (1:1,000; 10026-1-AP), Bcl-2-associated death promoter (BAD; 1:1,000; 10435-1-AP) and GAPDH (1:4,000; 60004-1-Ig) were purchased from Proteintech (Manchester, UK), while monoclonal antibodies against p-AKT (The-308; 1:800; 2118-1), p-AKT (Ser-473; 1:800; 2214-1), PI3K (1:1,000; 1593-S), PTEN (1:1,000; 5171-1), p-PTEN (1:1,000; 2134-1), Bcl-2 (1:1,000; 10026-1-AP), pro-caspase-9 (1:1,000; 1023-1) and cyclin B1 (1:2,000; 1495-1) were obtained from Abcam (Cambridge, UK), and monoclonal anti-cleaved-caspase-9 (1:1,000; sc-22182) was from Santa Cruz Biotechnology, Inc. (Shanghai, China).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 1. Evaluation of the mouse emphysema model. H&E staining of lung tissues from the (A) control, (B) emphysema, (C) PI3K inhibitor and (D) PDK1 inhibitor groups. Magnification, x400. (E) IL‑6 protein levels and (F) total cell count in BALF. Numbers of (G) neutrophils and (H) macrophages in BALF. (I) MLI and (J) DI were measured show the extent of airway remodelling in lung tissues. aP<0.05 vs. control. bP<0.05 vs. emphysema. BALF, bronchoalveolar lavage fluid; MLI, mean linear intercept; DI, destructive index; CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PI3K, phosphatidylinositol‑3‑kinase; PDK1, phosphoinositide dependent protein kinase 1.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Staining, Control, Cell Counting

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 3. Expression of LC3B protein in airway epithelial tissues. (A) Immunofluorescence staining for LC3BII in airway epithelial tissue. Magnification, x400. (B) Comparison of LC3B protein expression among the experimental groups. aP<0.05 vs. control. bP<0.05 vs. emphysema. CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PDK1, phosphoinositide dependent protein kinase 1; MFI, mean fluorescence intensity.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Expressing, Immunofluorescence, Staining, Comparison, Control, Fluorescence

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 2. Expression of PI3K, PDK1 and AKT proteins in the airway epithelial tissue. Immunohistochemical staining for (A) PI3K (B) PDK1 (C) AKT expression in the airway epithelial tissues. Magnification, x400. Quantification of (D) PI3K (E) PDK1 (F) AKT protein expression in the airway epithelial tissues in each group. aP<0.05 vs. control. bP<0.05 vs. emphysema. CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PDK1, phosphoinositide dependent protein kinase 1; AOD, average optical density.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Expressing, Immunohistochemical staining, Staining, Control

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 4. Expression of p16 protein in the airway epithelial tissues. (A) Immunofluorescence staining for the analysis of p16 protein expression in airway epithelial tissues. Magnification, x400. (B) Comparison of p16 protein expression among the experimental groups. aP<0.05 vs. control. bP<0.05 vs. emphysema. CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PDK1, phosphoinositide dependent protein kinase 1; p16, cyclin‑dependent kinase inhibitor 2A.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Expressing, Immunofluorescence, Staining, Comparison, Control

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 5. PI3K, PDK1, AKT, LC3B II and p16 protein expression levels. Western blotting for (A) PI3K, (B) PDK1, (C) AKT, (D) LC3B and (E) p16 expression in the airway epithelial tissues. GAPDH as an internal control. Semi‑quantification of PI3K, PDK1, AKT, LC3B and p16 protein expression in airway epithelial tissues in each group. aP<0.05 vs. control. bP<0.05 vs. emphysema. The protein expression levels were expressed as the ratio of band intensity for the target protein relative to that for the internal control GAPDH. Values are expressed as the mean ± standard deviation. CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PDK1, phosphoinositide dependent protein kinase 1; p16, cyclin‑dependent kinase inhibitor 2A.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Expressing, Western Blot, Control, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Glutamine deficiency promotes stemness and chemoresistance in tumor cells through DRP1-induced mitochondrial fragmentation

doi: 10.1007/s00018-021-03818-6

Figure Lengend Snippet: Cancer cells can survive and rearrange their metabolic phenotype in absence of glutamine. a Cell proliferation assay was done by counting cell number in 3 consecutive time point (24 h, 48 h, and 72 h) in glutamine-deprived condition (indicated as W/O GLN) in PA1 (n = 3) and HCT116 (n = 2) cells. b Histogram of cell cycle analysis and corresponding bar diagram of the histogram depicts that the cells were arrested in G0 phase upon glutamine deprivation in both PA1 and HCT116 (n = 3). c Apoptosis assay conducted in glutamine-deprived condition (24 h) and did not find any change in the live-cell population of PA1, and HCT116 (n = 3). d Red/green fluorescent population (%) was quantified for mitochondrial membrane potential using JC-1 dye in PA1, HCT116, and OAW42 upon 24 h of glutamine limiting condition, and presented in bar diagram (n = 3). e, f Extracellular flux analysis dictates that the cells became more glycolytic in nature while OXPHOS remained unchanged at 24 h time point in PA1 and OAW42 cells. g At 24 h of glutamine deprivation, glycolysis rate and glycolytic capacity increased in both PA1 and OAW42 cells. h Glucose uptake was increased in 24 h of glutamine deprivation in PA1 (n = 3). i Glucose oxidation through TCA cycle was increased in absence of glutamine at 24 h in PA1 (n = 3). j PDH phosphorylation increased in absence of glutamine at 24 h shown by Western blot in PA1 cells. k Glycostress assay kit was used to check the change in OCR after glucose injection in glutamine-deprived condition and silencing the PDHE1α in PA1 cells. l Heatmap of metabolites measured through NMR spectroscopy at 24 h of glutamine-starved condition in PA1. m Lactate level was increased in the medium when the cells are cultured in 24 h of glutamine limiting condition in PA1 (n = 3). Data are expressed in ± SEM, and statistical significance was calculated using two-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). NS non-significant

Article Snippet: The siRNA against DRP1 (Cat#sc-43732), xCT (Cat#sc-76933) and PDHE1α (Cat#sc-91064) (Santa Cruz Biotechnology; Dallas, Texas,United State) were used at 20 nM concentration along with lipofectamine 2000 (Cat#11668019, Invitrogen) in the cell lines.

Techniques: Proliferation Assay, Cell Cycle Assay, Apoptosis Assay, Membrane, Phospho-proteomics, Western Blot, Injection, Structural Proteomics, Cell Culture, Two Tailed Test