Journal: Molecular and Cellular Biology

Article Title: Regulation of Insulin Signaling by the Phosphatidylinositol 3,4,5-Triphosphate Phosphatase SKIP through the Scaffolding Function of Pak1

doi: 10.1128/MCB.00636-12

Figure Lengend Snippet: Insulin-dependent formation of a SKIP-Akt2 complex. (A) Induction of protein complex formation between SKIP, insulin receptor β, and Nck by insulin stimulation. C2C12 cells were transfected with the indicated constructs and stimulated with insulin for 0 or 30 min. Lysates from these cells were immunoprecipitated (I.P.) with anti-FLAG antibody. (B) Colocalization of endogenous SKIP and insulin receptor β in insulin-stimulated C2C12 cells. Cells were visualized by confocal microscopy. F-actin was visualized with Alexa Fluor 647-labeled phalloidin. Yellow indicates regions of colocalization of SKIP and insulin receptor β; white shows regions of colocalization of SKIP, insulin receptor β, and actin. Scale bar, 20 μm. (C) Insulin-dependent formation of a SKIP-Akt2 complex. C2C12 cells were transfected with 3× FLAG-tagged SKIP WT and then stimulated with insulin for the indicated times. Lysates were immunoprecipitated with anti-FLAG antibodies. (D) Pak1 mediates complex formation between endogenous SKIP and Akt2. C2C12 cells were transfected with control or Pak1 siRNA. Relative expression of Pak1 in these cells is shown (upper panels). These cells were stimulated with insulin for 0 or 30 min. Lysates were immunoprecipitated with anti-SKIP antibody, and the immunoprecipitates were detected with anti-Akt2, -PDK1, or -Pak1 antibodies. The amounts of Akt2 and PDK1 immunoprecipitated with SKIP are shown (lower panels). Results are presented as the means ± SEMs of 5 independent experiments. *, P < 0.05; **, P < 0.01. (E) Pak1 predominantly associated with Akt2 in C2C12 cells. C2C12 cells were serum deprived for 24 h and then stimulated with insulin for the indicated times. Lysates were immunoprecipitated with anti-rabbit IgG and anti-Pak1 antibody. Immunoprecipitates were subjected to Western blot analysis.

Article Snippet: Anti-insulin receptor β, Rac1, and PDK1 antibodies were purchased from BD Biosciences (Franklin Lakes, NJ).

Techniques: Transfection, Construct, Immunoprecipitation, Confocal Microscopy, Labeling, Expressing, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Regulation of Insulin Signaling by the Phosphatidylinositol 3,4,5-Triphosphate Phosphatase SKIP through the Scaffolding Function of Pak1

doi: 10.1128/MCB.00636-12

Figure Lengend Snippet: Dissociation of Akt2 from the Pak1 complex, as mediated by the PIP3 phosphatase activity of SKIP. (A) Decreased Rac1 activity in insulin-stimulated C2C12 cells caused by expression of SKIP WT but not the D310G mutant. Results are presented as the means ± SDs of 3 independent experiments. **, P < 0.01. (B) Suppression of Pak1 activity caused by expression of SKIP WT but not the D310G mutant. Results are presented as the means ± SDs of 3 independent experiments. *, P < 0.05. (C) Increased Rac1 activity caused by the silencing of SKIP. Results are presented as the means ± SDs of 3 independent experiments. *, P < 0.05. (D) Increased Pak1 activity, measured as insulin-induced phosphorylation of Bad at Ser-112, caused by SKIP attenuation in C2C12 cells. Results are presented as the means ± SDs of 3 independent experiments. **, P < 0.01. (E) Decreased binding of Akt2 and PDK1 to Pak1 caused by the PIP3 phosphatase activity of SKIP. (F) Suppression of Akt2 activity caused by expression of SKIP WT but not the D310G mutant. Results are presented as the means ± SDs of 3 independent experiments. *, P < 0.05.

Article Snippet: Anti-insulin receptor β, Rac1, and PDK1 antibodies were purchased from BD Biosciences (Franklin Lakes, NJ).

Techniques: Activity Assay, Expressing, Mutagenesis, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Regulation of Insulin Signaling by the Phosphatidylinositol 3,4,5-Triphosphate Phosphatase SKIP through the Scaffolding Function of Pak1

doi: 10.1128/MCB.00636-12

Figure Lengend Snippet: Working model of SKIP regulation of insulin signaling. (A) Under resting conditions, SKIP is localized to the ER. (B) Insulin-dependent PIP3 generation and Rac1-dependent Pak1 activation. Pak1 activation triggers its recruitment to the insulin receptor complex and subsequent complex formation with Akt2 and PDK1. Activated signaling molecules are highlighted in red. (C) Insulin induces translocation of SKIP to the plasma membrane, where SKIP binds to active Pak1 (the open form). Formation of a complex including SKIP, Pak1, Akt2, and Rac1 is induced at the membrane ruffles. (D) SKIP hydrolyzes PIP3 bound to Akt2. Localization of SKIP in proximity to Akt2 facilitates its inactivation. Signaling molecules inactivated by SKIP are indicated in blue. (E) SKIP negatively regulates Rac1 and Pak1 activity, leading to the inactive conformation of Pak1. Dissociation of Akt2 and PDK1 from inactive Pak1 terminates insulin signaling.

Article Snippet: Anti-insulin receptor β, Rac1, and PDK1 antibodies were purchased from BD Biosciences (Franklin Lakes, NJ).

Techniques: Activation Assay, Translocation Assay, Activity 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 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: Cell Reports Medicine

Article Title: Arachidonic acid released by PIK3CA mutant tumor cells triggers malignant transformation of colonic epithelium by inducing chromatin remodeling

doi: 10.1016/j.xcrm.2024.101510

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal antibody anti-PDK1 , Cell Signaling Technology , Cat# 5662; RRID: AB_10839264.

Techniques: Microarray, Recombinant, Cloning, Mutagenesis, Immunohistochemistry, Protein Purification, In Vitro, Sequencing, Methylation

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: Cancer Science

Article Title: WDR54 exerts oncogenic roles in T‐cell acute lymphoblastic leukemia

doi: 10.1111/cas.15872

Figure Lengend Snippet: WDR54 modulates PDPK1, AKT, ERK, cleaved caspase‐3, cleaved caspase‐9, Bcl‐2, and Bcl‐xL signal pathways in T‐cell acute lymphoblastic leukemia (T‐ALL) cell lines. Cell lysates from shC or shWDR54 cells at 3 days post‐infection were analyzed. (A) The protein expression of p‐AKT, AKT, p‐ERK, and ERK in Molt4 (left) and Jurkat (right) cells was detected by immunoblots, respectively. (B) Analysis of cleaved caspase‐3, cleaved caspase‐9, Bcl‐2, and Bcl‐xL was performed by immunoblots. (C) Heatmap analysis of RNAseq expression data showing differentially expressed genes in Jurkat cells ( p < 0.05). (D) The mRNA level of PDPK1 was detected by real‐time PCR in Molt4 cells (left) and Jurkat cells (right). (E) The protein expression of PDPK1 in Molt4 cells (left) and Jurkat cells (right). *** p < 0.001.

Article Snippet: Primary antibodies used in the experiments include β‐actin (1:50000, AC026, ABclonal), WDR54 (1:1000, HPA043257, Sigma), AKT (1:1000, 9272, Cell Signaling Technology), p‐AKT (1:1000, 4060, Cell Signaling Technology), ERK (1:1000, 4370, Cell Signaling Technology), p‐ERK (1:1000, 4695, Cell Signaling Technology), Bcl‐2 (1:1000, 3498, Cell Signaling Technology), Bcl‐xL (1:1000, ab98143, Abcam), cleaved Caspase‐3 (1:1000, 9664, Cell Signaling Technology), Caspase‐9 (1:1000, 9502, Cell Signaling Technology), GAPDH (1:5000, 60,004‐1‐Ig, Proteintech), and PDPK1 (1:1000, 17,086‐1‐AP, Proteintech).

Techniques: Infection, Expressing, Western Blot, Real-time Polymerase Chain Reaction