Journal: International Journal of Molecular Sciences

Article Title: Ilimaquinone Induces the Apoptotic Cell Death of Cancer Cells by Reducing Pyruvate Dehydrogenase Kinase 1 Activity

doi: 10.3390/ijms21176021

Figure Lengend Snippet: IQ reduced PDK activity and promoted OxPhos in A549 cells. The A549 cells were treated with IQ for 4 h. ( A , B ) The levels of phosphorylated PDHA1 ( A ) and PDK1–4 ( B ) were analyzed via Western blotting. (( A ), lower panel ) The bar graph from three independent experiments (( A ), upper panel ) is shown; PDHA and GAPDH were used as loading controls. ( C ) Lactate production after IQ treatment was measured using a lactate fluorometric assay kit. ( D ) The cells were treated with IQ, and the O 2 consumption rate was measured using a commercially available Oxygen Consumption Rate Assay Kit. ( E ) The cells were treated with the indicated concentrations of IQ for 24 h. The viability of these cells was measured using the MTT assay. All experiments except for panel B were performed at least three times, and data are shown as the mean ± SD. Statistical analyses were performed using Student’s t -test. *, p < 0.01 **, p < 0.05 and ***, p < 0.001 compared with the control.

Article Snippet: The antibody against PDK1 (#ADI-KAP-PK112) was purchased from Enzo Life Sciences (Farmingdale, NY, USA).

Techniques: Activity Assay, Western Blot, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Ilimaquinone Induces the Apoptotic Cell Death of Cancer Cells by Reducing Pyruvate Dehydrogenase Kinase 1 Activity

doi: 10.3390/ijms21176021

Figure Lengend Snippet: IQ interacted with the ATP binding pocket of PDK1. ( A ) The ITC analysis results of PDK1 and IQ are shown. IQ was titrated into PDK1 solutions, and the measured Kd values are shown. ( B ) IQ binds to the ATP binding pocket of PDK1. The modeled structure of PDK1 with IQ is shown as a ribbon diagram. The interaction residues between PDK1 and IQ are shown. ( C ) The ATP binding assay results of PDK1 and IQ are shown. The binding of ATP to PDK1 in the presence of IQ was determined using α 32 -labeled ATP. Non-bound α- 32 P-GTP was washed out, and the radioactivity was examined using a scintillation counter. Non-labeled ATP was used as a competitor. Data are presented as the mean ± SD. Statistical analyses were performed using Student’s t -test. *** p < 0.001 compared with the control under α 32 -labeled ATP (second column). ( D ) The PDC subunits and PDK binding were analyzed.

Article Snippet: The antibody against PDK1 (#ADI-KAP-PK112) was purchased from Enzo Life Sciences (Farmingdale, NY, USA).

Techniques: Binding Assay, Labeling, Radioactivity

Journal: International Journal of Biological Sciences

Article Title: Triptolide Suppresses Glomerular Mesangial Cell Proliferation in Diabetic Nephropathy Is Associated with Inhibition of PDK1/Akt/mTOR Pathway

doi: 10.7150/ijbs.20485

Figure Lengend Snippet: The effect of TP on PDK1 expression in vivo and in vitro . (A) The expression of PDK1 in the kidney of HFD/STZ-induced diabetic rats. (B) Quantification of results in A. (C) The expression of PDK1 in HRMCs treated for 72 h. (D) Quantification of results in C. Data were reported as mean ± S.D.. * P < 0.05

Article Snippet: The primary antibodies and their dilutions used were as follows: PDK1 (1:1000, Cell Signaling Technology, USA), total-Akt (1:2000, Cell Signaling Technology, USA), phosphorylation-Akt (1:1000, Cell Signaling Technology, USA), total-mTOR (1:2000, Cell Signaling Technology, USA), phosphorylation-mTOR (1:1000, Cell Signaling Technology, USA), Ki-67 and PCNA (all diluted as 1:1000, Proteintech, USA).

Techniques: Expressing, In Vivo, In Vitro

Journal: International Journal of Biological Sciences

Article Title: Triptolide Suppresses Glomerular Mesangial Cell Proliferation in Diabetic Nephropathy Is Associated with Inhibition of PDK1/Akt/mTOR Pathway

doi: 10.7150/ijbs.20485

Figure Lengend Snippet: PDK1 activator reversed the effect of TP on mesangial cell proliferation. (A) MTT assay in the cells treated with HG for different times. * P < 0.05 vs. Control group, # P < 0.05 vs. HG+TP group. (B and C) Flow cytometry analysis of cell cycle in the HRMCs treated for 72 h. (D) Protein expression of PDK1/Akt/mTOR pathway in HRMCs treated for 72 h. (E) Protein expression of Ki-67 and PCNA in HRMCs treated for 72 h. (F) Immunofluorescence images of Ki-67 and PCNA. The scale bar represents 10 μm. Data were reported as mean ± S.D.. * P < 0.05; ns represents no significance

Article Snippet: The primary antibodies and their dilutions used were as follows: PDK1 (1:1000, Cell Signaling Technology, USA), total-Akt (1:2000, Cell Signaling Technology, USA), phosphorylation-Akt (1:1000, Cell Signaling Technology, USA), total-mTOR (1:2000, Cell Signaling Technology, USA), phosphorylation-mTOR (1:1000, Cell Signaling Technology, USA), Ki-67 and PCNA (all diluted as 1:1000, Proteintech, USA).

Techniques: MTT Assay, Control, Flow Cytometry, Expressing, Immunofluorescence

Journal: Oncogenesis

Article Title: TCRP1 promotes NIH/3T3 cell transformation by over-activating PDK1 and AKT1

doi: 10.1038/oncsis.2017.18

Figure Lengend Snippet: TCRP1 directly interacts with PDK1 and activates PDK1/AKT signaling.( a ) The effects of TCRP1 on the expression of PI3K/Akt and its downstream molecules. ( b ) 293T and NIH/3T3 cells were co-transfected with EGFP-TCRP1 and pDsred-PDK1, or EGFP-TCRP1 and pDsred-AKT1, the direct interaction between TCRP1 and PDK1, or TCRP1 and AKT1, were determined by forster resonance energy transfer (FRET) assay. Cells co-transfected with EGFP-PDK1 plasmids, and pDsred-AKT1 was used as positive control. ( c ) Total protein extracts of NIH/3T3 cells were subjected to IP using TCRP1 antibody or control IgG, followed by western blotting (WB) with PDK1 antibody (upper panel). Reciprocal IP was done using PDK1 antibody or control IgG, followed by WB with the TCRP1 antibody (lower panel).

Article Snippet: Antibody against PDK1 (#sc-7140) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Förster Resonance Energy Transfer, Positive Control, Control, Western Blot

Journal: Oncogenesis

Article Title: TCRP1 promotes NIH/3T3 cell transformation by over-activating PDK1 and AKT1

doi: 10.1038/oncsis.2017.18

Figure Lengend Snippet: The PDK1-binding domain of TCRP1 is R93-S107 and T109-A124.( a ) Motif analysis of TCRP1 was processed in Scansite database ( http://scansite.mit.edu/ ), and showed that the 93–107 amino-acid and 109–124 amino-acid sequences in the TCRP1 were PDK1-binding motif (upper panel). Two vectors including the two TCRP1 mutant fragments were constructed named pEGFP-TCRP1/mut1 and pEGFP-TCRP1/mut2, respectively (lower panel). ( b ) NIH/3T3 cells were co-transfected with EGFP-TCRP1/mut1 and pDsred-PDK1, or EGFP-TCRP1/mut2 and pDsred-PDK1, the direct interaction between TCRP1/muts and PDK1 was determined by forster resonance energy transfer (FRET) assay. ( c ) NIH/3T3 cells were transfected with pEGFP-TCRP1/mut1, or pEGFP-TCRP1/mut2, total protein extracts were subjected to IP using TCRP1 antibody or control IgG, followed by WB with PDK1 antibody. Reciprocal IP was done using PDK1 antibody or control IgG, followed by WB with the TCRP1 antibody.

Article Snippet: Antibody against PDK1 (#sc-7140) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Binding Assay, Mutagenesis, Construct, Transfection, Förster Resonance Energy Transfer, Control

Journal: Oncogenesis

Article Title: TCRP1 promotes NIH/3T3 cell transformation by over-activating PDK1 and AKT1

doi: 10.1038/oncsis.2017.18

Figure Lengend Snippet: Inhibition of PDK1 reversed TCRP1-mediated cell transformation of NIH/3T3 cells. ( a , b ) NIH/3T3/TCRP1 cells or NIH/3T3/TCRP1 primary cells were treated with PDK1 inhibitor OSU-03012, or TCRP1 siRNA, the expression levels of p-PDK1, PDK1 and cyclin D1 were measured by western blotting ( a ); cell viability were measured by MTS assay ( b ). ( c , d ) NIH/3T3/TCRP1 cells or NIH/3T3/TCRP1 primary cells were treated with PDK1 inhibitor OSU-03012, clonogenic capacity were measured by plate cloning assay and soft agar cloning assay ( c ). Cells were fixed in ethanol, and stained with propidium iodide, and then DNA contents were determined by flow cytometry ( d ). The percentage of cells in each phase of the cell cycle (G1, S and G2/M) was indicated. Experiments were repeated three times. * P <0.05.

Article Snippet: Antibody against PDK1 (#sc-7140) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Transformation Assay, Expressing, Western Blot, MTS Assay, Cloning, Staining, Flow Cytometry

Journal: Oncogenesis

Article Title: TCRP1 promotes NIH/3T3 cell transformation by over-activating PDK1 and AKT1

doi: 10.1038/oncsis.2017.18

Figure Lengend Snippet: TCRP1 was highly expressed in many tumors and was positively correlated with expression of p-PDK1 and p-AKT. ( a ) Expression of TCRP1 was detected with PCR chip in a variety of tumors and corresponding normal tissue. ( b ) Expression of TCRP1, p-PDK1 and p-AKT1 were detected by immunohistochemistry in lung cancer and glioma. ( c ) Scatter plot showed relative abundance of TCRP1, p-PDK1 and p-AKT1 in lung cancer and glioma; * P <0.05 vs TCRP1. ( d ) The correlation trend of expression of TCRP1, p-PDK1 and p-AKT1 in glioma and lung cancer.

Article Snippet: Antibody against PDK1 (#sc-7140) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Immunohistochemistry

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) The landscape of PDK1-related clinicopathological features in osteosarcoma from the UCSCXena database. No statistically significant differences in PDK1 expression were observed among the following subgroups: (B) Age group (>18 vs. ≤ 18 years), (C) Gender group,(D) Metastasis status group, and (E) 3-year survival status group. Statistical significance was assessed using an unpaired t test.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Expressing

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) PDK1 was enriched in the deceased group of osteosarcoma patients in the UCSCXena databases. The significance of the difference was tested by unpaired t test. (B) The receiver-operating characteristic (ROC) curve showed the high-expression specificity of PDK1 in deceased group of osteosarcoma patients. AUC, area under the curve.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Expressing

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) AUC curve shows that the precision of this model is good. (B) Kaplan-Meier survival curves illustrate the increasing expression of PDK1 was negatively correlated with prognosis of patients with osteosarcoma. (C) The expression of PDK1 was negatively correlated with prognosis in non-metastic osteosarcoma group. (D)There was no significant correlation between PDK1 expression and prognosis in metastatic osteosarcoma group.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Expressing

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) Top 50 genes most negatively associated with PDK1 are shown in a heatmap. (B) Top 50 genes most positively associated with PDK1 are shown in a heatmap. (C) GO functional annotation analysis showed that PDK1 played a key role in biological processes such as metabolism and cell proliferation. (D) KEGG enrichment analysis showed that positive related genes with PDK1 were mostly enriched in cell metabolism and proliferation pathways, while negative related genes were enriched in immune-related signaling pathways. (E) Reactome analysis of significant gene pathways negatively correlated with PDK1. (F) Reactome analysis of significant gene pathways positively correlated with PDK1.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Functional Assay, Protein-Protein interactions

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) Connected dot plot comparing TP53 expression with PDK1 expression between the PDK1-high and PDK1-low groups. (B) Connected dot plot comparing CDKN2A expression with PDK1 expression between the PDK1-high and PDK1-low groups. (C) Connected dot plot comparing MDM2 expression with PDK1 expression between the PDK1-high and PDK1-low groups. (D) Connected dot plot comparing RB1 expression with PDK1 expression between the PDK1-high and PDK1-low groups.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Expressing

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) Distributions of PDK1 expression levels are displayed using box plots. The statistical significance computed by the Wilcoxon test is annotated by the number of stars. (B) Overall survival analysis the prognostic relationship of PDK1 expression in different types of cancer by Cox proportional hazards regression model. (C) DFI analysis the prognostic relationship of PDK1 expression in different types of cancer by Cox proportional hazards regression model.(D-I) Pan-cancer Kaplan-Meier overall survival of PDK1 in indicated tumor types from TCGA database. The median value of PDK1 in each tumor was taken as the percentile (50%) of PDK1 expression.(J-L) Pan-cancer differential expression of PDK1 in WHO stages in indicatedumor types from TCGA database. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Expressing, Quantitative Proteomics

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) PDK1 expression and clinical features. (B)The calibration of the nomogram at 1, 3, and 5 years in the training cohort.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Expressing

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) Comparison of PDK1 mRNA expression between U-2 OS, 143B, MG63 and hFOB1.19 by qRT-PCR(n = 3). (B) Comparison of PDK1 protein expression between U-2 OS, 143B, MG63 and hFOB1.19 by western blotting(n = 3). (C) Analysis of PDK siRNA transfection efficiency in 143B cells by qRT-PCR(n = 3). (D) Detection of 143B viability at 24 hrs, 48 hrs and 72 hrs after silencing PDK1 by CCK-8 analysis(n = 3). (E) The effect of PDK1 siRNA on colony forming ability of 143B was detected by clone formation assay(n = 1). (F) Flow cytometry was used to detect the effect of PDK1 siRNA group and control group on apoptosis of 143B cells(n = 3). (G) Flow cytometry was used to detect the effect of PDK1 siRNA group and control group on cell cycle of 143B cells(n = 3). (H) Analysis of lactate content: The lactate produced during the glycolytic metabolism of 143B cells in the silenced PDK1 group and the control group(n = 3). (I) Analysis of ATP content: The ATP produced during the glycolytic metabolism of 143B cells in the silenced PDK1 group and the control group(n = 3). (J) Analysis of C6P content: The C6P produced during the glycolytic metabolism of 143B cells in the silenced PDK1 group and the control group(n = 3). (K) Analysis of glucose content: The glucose content during the glycolytic metabolism of 143B cells in the silenced PDK1 group and the control group(n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Comparison, Expressing, Quantitative RT-PCR, Western Blot, Transfection, CCK-8 Assay, Tube Formation Assay, Flow Cytometry, Control, Produced

Journal: Molecular medicine reports

Article Title: Combination of Taxol® and dichloroacetate results in synergistically inhibitory effects on Taxol-resistant oral cancer cells under hypoxia.

doi: 10.3892/mmr.2014.3080

Figure Lengend Snippet: Figure 2. Hypoxia and Taxol treatments induce PDK1 mRNA and protein expression in OECM-1 human oral squamous cell carcinoma cells. (A) OECM‑1 cells were incubated in 2% oxygen for 24 h, after which cells were collected and whole cell lysates were subjected to western blot analysis. β‑actin was used as a loading control. The total mRNA of the cells, which had been cultured in both normoxic and hypoxic conditions, was isolated and used for cDNA synthesis and quantitative PCR. (B) OECM‑1 cells were treated with Taxol at 50 and 100 nM under normoxic conditions for 24 h. The cells were collected and the whole cell lysates were subjected to western blotting. β‑actin was used as a loading control . The total mRNA of Taxol‑treated cells was isolated followed by cDNA synthesis and quantitative PCR. The data represent the means of the three independent experiments ± standard error. **P<0.01; ***P<0.001. Ctrl, control; PDK1, pyruvate dehydrogenase kinase 1; PCR, polymerase chain reaction; mRNA, messenger RNA.

Article Snippet: A rabbit monoclonal antibody against PDK1 was used at a 1:1,000 dilution(#3820; Cell Signaling Technology, Danvers, MD, USA); a β-Actin antibody was used as a loading control, at a 1:2,000 dilution (sc-1616; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Western Blot, Control, Cell Culture, Isolation, cDNA Synthesis, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: Combination of Taxol® and dichloroacetate results in synergistically inhibitory effects on Taxol-resistant oral cancer cells under hypoxia.

doi: 10.3892/mmr.2014.3080

Figure Lengend Snippet: Figure 3. Taxol resistant cells exhibit upregulated PDK1 mRNA and protein expression levels. (A) The Taxol‑resistant cells were generated from OECM‑1 human oral cancer cells. Taxol-sensitive and resistant cells were treated with Taxol at 5 and 10 µM for 24 h, followed by MTT assay to measure cell viability. (B) The Taxol-sensitive and resistant cells were collected and the whole cell lysates were subjected to western blotting. β‑actin was used as a loading control. (C) Total mRNA from Taxol-sensitive and resistant cells was isolated, followed by cDNA synthesis and quantitative PCR. The data represent the means of the three independent experiments ± standard error. *P<0.05; **P<0.01. PDK1, pyruvate dehydrogenase kinase 1; Ctrl, control; mRNA, messenger RNA; OCEM-1, human oral squamous cell carcinoma cell line; PCR, polymerase chain reaction; S, sensitive; R, resistant.

Article Snippet: A rabbit monoclonal antibody against PDK1 was used at a 1:1,000 dilution(#3820; Cell Signaling Technology, Danvers, MD, USA); a β-Actin antibody was used as a loading control, at a 1:2,000 dilution (sc-1616; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Generated, MTT Assay, Western Blot, Control, Isolation, cDNA Synthesis, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: Combination of Taxol® and dichloroacetate results in synergistically inhibitory effects on Taxol-resistant oral cancer cells under hypoxia.

doi: 10.3892/mmr.2014.3080

Figure Lengend Snippet: Figure 4. Combination treatment of DCA and Taxol exhibits a synergistic inhibitory effect on Taxol-resistant cells. (A) DCA treatments were shown to inhibit PDK1 protein expression, as compared with the control. OECM‑1 human oral cancer cells were treated with DCA at 5 and 10 mM for 24 h followed by western blotting. β‑actin was used as a loading control. (B) Taxol‑sensitive cells, cultured in normoxic conditions, were not affected by the enhanced inhibition resulting from the combination of DCA and Taxol. Taxol‑resistant cells showed synergistic effects from the combination of DCA and Taxol treatments. Under hypoxic conditions, the combination of DCA and Taxol produced synergistic effects on both Taxol‑sensitive and resistant cells. The data represent the means of the three independent experi ments ± standard error. PDK1, pyruvate dehydrogenase kinase 1; DCA, dichloroacetate; Ctrl, control.

Article Snippet: A rabbit monoclonal antibody against PDK1 was used at a 1:1,000 dilution(#3820; Cell Signaling Technology, Danvers, MD, USA); a β-Actin antibody was used as a loading control, at a 1:2,000 dilution (sc-1616; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Control, Western Blot, Cell Culture, Inhibition, Produced