Journal: Cell Division

Article Title: DNMT3B blocks TAL1-mediated PKM2 transcriptional repression to promote non-small cell lung cancer progression through inducing glycolysis

doi: 10.1186/s13008-025-00168-8

Figure Lengend Snippet: TAL1 inhibits NSCLC progression by blocking the glycolytic pathway through inhibition of PKM2. A Colony formation assay detected the colony-forming of A549 and H60 cells after 14 days of culture. B MTT analyzed the proliferation ability. C EdU assay verified the proliferation ability. D Wound healing assay detected migration. E Transwell assay detected the invasion ability. F TUNEL assay detected the apoptosis rate. G Cellular metabolism assay detected changes in lactate levels. H WB assay detected the protein levels of glycolysis-related proteins GLUT1, LDHA, and PDK1. Comparisons were made using unpaired t-tests or two-way ANOVA. ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The tissues were incubated with primary antibodies against GLUT1 (1:50, A6982, ABclonal), LDHA (1:200, 3582, CellSignaling), PDK1 (1:500, NBP2-44132, Novus Biologicals), Ki67 (1:1000, ab92742, Abcam), and Cleaved-Caspase3 (1:400, MAB835, Novus Biologicals) at 4 °C overnight and with goat anti-rabbit secondary antibody coupled to HRP (1:1000, ab6721, Abcam) for 30 min.

Techniques: Blocking Assay, Inhibition, Colony Assay, EdU Assay, Wound Healing Assay, Migration, Transwell Assay, TUNEL Assay

Journal: Cell Division

Article Title: DNMT3B blocks TAL1-mediated PKM2 transcriptional repression to promote non-small cell lung cancer progression through inducing glycolysis

doi: 10.1186/s13008-025-00168-8

Figure Lengend Snippet: TAL1 blocks the glycolytic pathway by inhibiting PKM2 to slow NSCLC progression. A Tumor images and tumor weight of mice. B The tumor volume of mice was measured periodically. C WB detected the expression of TAL1 and PKM2 in the tumor tissues of mice. D IHC detected the expression of glycolysis-related proteins (GLUT1, LDHA, and PDK1), Ki67, and Cleaved-Caspase3 in the tumors of mice. E TUNEL detected apoptosis in tumor tissues of mice. Animal experiments had five mice in each group. Comparisons were made among multiple groups using one-way or two-way ANOVA. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant

Article Snippet: The tissues were incubated with primary antibodies against GLUT1 (1:50, A6982, ABclonal), LDHA (1:200, 3582, CellSignaling), PDK1 (1:500, NBP2-44132, Novus Biologicals), Ki67 (1:1000, ab92742, Abcam), and Cleaved-Caspase3 (1:400, MAB835, Novus Biologicals) at 4 °C overnight and with goat anti-rabbit secondary antibody coupled to HRP (1:1000, ab6721, Abcam) for 30 min.

Techniques: Expressing, TUNEL Assay

Journal: Cell Division

Article Title: DNMT3B blocks TAL1-mediated PKM2 transcriptional repression to promote non-small cell lung cancer progression through inducing glycolysis

doi: 10.1186/s13008-025-00168-8

Figure Lengend Snippet: DNMT3B activates the glycolytic pathway by inhibiting TAL1 expression. A Colony formation assay to detect the proliferation of A549 and H460 cells after 14 days of culture. B EdU assay verified the proliferative ability of A549 and H460 cells. C , D The Transwell assay detected the migratory and invasive ability of A549 and H460 cells. E TUNEL assay detected apoptotic rate. F Cell metabolism assay detected changes in lactate levels. G WB detection of protein levels of glycolysis-related proteins GLUT1, LDHA, and PDK1. Comparisons were made among multiple groups using one-way or two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The tissues were incubated with primary antibodies against GLUT1 (1:50, A6982, ABclonal), LDHA (1:200, 3582, CellSignaling), PDK1 (1:500, NBP2-44132, Novus Biologicals), Ki67 (1:1000, ab92742, Abcam), and Cleaved-Caspase3 (1:400, MAB835, Novus Biologicals) at 4 °C overnight and with goat anti-rabbit secondary antibody coupled to HRP (1:1000, ab6721, Abcam) for 30 min.

Techniques: Expressing, Colony Assay, EdU Assay, Transwell Assay, TUNEL Assay

Journal: Cell Division

Article Title: DNMT3B blocks TAL1-mediated PKM2 transcriptional repression to promote non-small cell lung cancer progression through inducing glycolysis

doi: 10.1186/s13008-025-00168-8

Figure Lengend Snippet: DNMT3B affects NSCLC progression in vivo by regulating the TAL1/PKM2 axis. A Tumor images and tumor weight of mice. B The tumor volume of mice was measured periodically. C WB detection of DNMT3B, TAL1, and PKM2 protein expression in tumor tissues of mice. D IHC detection of glycolysis-related proteins (GLUT1, LDHA, and PDK1), Ki67, and Cleaved-Caspase3 in the tumors of mice. E TUNEL detection of apoptosis in tumor tissues. Animal experiments had five mice in each group. One-way or two-way ANOVA was used for single-factor comparisons among multiple groups. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant

Article Snippet: The tissues were incubated with primary antibodies against GLUT1 (1:50, A6982, ABclonal), LDHA (1:200, 3582, CellSignaling), PDK1 (1:500, NBP2-44132, Novus Biologicals), Ki67 (1:1000, ab92742, Abcam), and Cleaved-Caspase3 (1:400, MAB835, Novus Biologicals) at 4 °C overnight and with goat anti-rabbit secondary antibody coupled to HRP (1:1000, ab6721, Abcam) for 30 min.

Techniques: In Vivo, Expressing, TUNEL Assay

Journal: The FASEB Journal

Article Title: Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol‐dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation

doi: 10.1096/fj.201600330rr

Figure Lengend Snippet: Figure 5. Sequence coverage of phosphorylated hBVR by proteolysis and mass spectrometry. Products of kinase reaction that included Akt1 and his6-hBVR (purified from E. coli) were resolved by gel electrophoresis. Three identical samples, each containing 2 mg of protein, were digested with trypsin, chymotrypsin, or Arg-C protease. Products were identified by mass spectrometry. Extent of sequence coverage by each digest is indicated by underlining; serine residues identified as phosphorylation sites are indicated by dots above sequence. Red shading indicates Akt binding/phosphorylation site as predicted by computational analysis, and blue shading indicates RFxFPxFS PDK1 binding motif.

Article Snippet: The open reading frame for PDK1 was amplified from a clone (pDONR223-PDPK1, a gift from W. Hahn and D. Root (Addgene plasmid 23801), and was cloned into pcDNA3.

Techniques: Sequencing, Mass Spectrometry, Nucleic Acid Electrophoresis, Phospho-proteomics, Binding Assay

Journal: The FASEB Journal

Article Title: Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol‐dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation

doi: 10.1096/fj.201600330rr

Figure Lengend Snippet: Figure 7. A) Coimmunoprecipitation of PDK1 and hBVR. Cells were cotransfected with PDK1 expression plasmid and pcDNA- HA-hBVR. Lysates were prepared from cells with or without stimulation with 40 ng/ml IGF-1 and immunoprecipitated with anti- HA antibody. Immunoprecipitates were analyzed by Western blot test; blot was sequentially probed with antibodies to PDK1 and hBVR. Expression of PDK1 and hBVR in lysates was verified by Western blot test. B) PDK1, Akt1, and hBVR coimmunoprecipitate in IGF-1 stimulated cells. Lysates prepared from cells cotransfected with pcDNA-HA-hBVR and pcDNA-Akt1 and stimulated with 40 ng/ml IGF-1 or 50 ng/ml TNF-a were immunoprecipitated with anti-HA antibodies. Immunoprecipitates were analyzed by Western blot test, which was sequentially probed with antibodies to PDK1, Akt1, and hBVR. Overexpression of Akt1 and HA- hBVR, and presence of PDK1 were verified by Western blot test of cell lysates. C) hBVR consensus PDK1 binding site is important in PDK1 and Akt1 interaction. Cells were cotransfected with PDK1 expression plasmid with or without pcDNA-HA-hBVR or pcDNA-HA-hBVR C-Box (RFGFPAFS to RVGAPAVS) mutant. Immunoprecipitates obtained with Akt1 antibody were analyzed for PDK1 as in (B); equality of loading was confirmed by probing blot with anti-Akt1 antibody. Overexpression of proteins was verified by Western blot test of cell lysates.

Article Snippet: The open reading frame for PDK1 was amplified from a clone (pDONR223-PDPK1, a gift from W. Hahn and D. Root (Addgene plasmid 23801), and was cloned into pcDNA3.

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Over Expression, Binding Assay, Mutagenesis

Journal: The FASEB Journal

Article Title: Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol‐dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation

doi: 10.1096/fj.201600330rr

Figure Lengend Snippet: Figure 8. A) Presence of hBVR decreases GSK3a/b activity in cell. Cells were transfected with either pcDNA-HA-hBVR or with siBVR. Lysates prepared from cells were assayed for GSK3a/b activity using peptide substrate from glycogen synthase, as described in text. **P , 0.01, ***P , 0.001. Western blots of lysates were probed with antibodies to phospho-GSK3b, GSK3a, GSK3b, hBVR, and anti–tubulin. B) sihBVR treatment prevents formation of hBVR/PDK1/Akt1 complex. Immunoprecipitates were analyzed by Western blot test, which was sequentially probed with PDK1 and Akt1 antibodies. Observed pattern of PDK1 is consistent with presence of splice variants that are recognized by antibody. C) GST pulldown shows association of GSK3b from insulin-treated cells with hBVR. Cells were transfected with pcDNA-GSK3b and subsequently treated with insulin or left untreated. Lysates were analyzed by GST pulldown using GST-hBVR, followed by Western blot analysis. Blots were probed with antibodies to GSK3b followed by anti-hBVR. Overexpression of GSK3b was verified by Western blot test. D) Phosphorylation of FoxO3 in response to IGF-1 is mediated by hBVR. Cells were transfected with pcDNA-HA-hBVR or with empty vector. After 24 h, transfected cells were treated with 40 ng/ml IGF-1 for 1 or 4 h or were left untreated. Phosphorylated FoxO3C and FoxO3A were detected by Western blot test, which was then probed with antibody to FoxO3A. Densitometry was used to normalize phospho- FoxO3 signals.

Article Snippet: The open reading frame for PDK1 was amplified from a clone (pDONR223-PDPK1, a gift from W. Hahn and D. Root (Addgene plasmid 23801), and was cloned into pcDNA3.

Techniques: Activity Assay, Transfection, Western Blot, Over Expression, Phospho-proteomics, Plasmid Preparation

Journal: The Journal of biological chemistry

Article Title: Pyruvate dehydrogenase kinase 1 controls triacylglycerol hydrolysis in cardiomyocytes.

doi: 10.1016/j.jbc.2025.108398

Figure Lengend Snippet: Figure 1. Effect of PDK1 suppression on PDH activity. A, PDK protein abundance measured by LC-MS/MS based proteomics respectively in control and Pdk1 knockdown (KD) differentiated H9c2 cardiomyocytes. B, schematic of experimental design showing differentiation of shRNA transduced H9c2 myoblasts for 10 days in 1% fetal bovine serum and 1 mM trans-retinoic acid for 10 days followed by pre-treatment with 375 mM palmitic acid in complete growth medium for 18 h and subsequent nutrient deprivation in low glucose, serum-free media. C, total PDH activity, (D) Total PDH abundance were assessed in control and Pdk1 knockdown cells following palmitic acid treatment and nutrient deprivation. Data are shown as mean ± SEM and were analyzed by either a Two-way ANOVA followed by a post hoc Tukey multiple comparison test (C and D) or multiple t test with Bonferroni-Dunn correction (A). For (C), ANOVA showed (F and p values) of (3.1 and 0.0906), (1.6 and 0.2118), (0.4 and 0.526) for interaction, row (nutrient availability) and column (cell lines) factors respectively. For (D), ANOVA showed (F and p) values of (6.523 and 0.0340), (1.416 and 0.2682), (9.783 and 0.0141) for interaction, row (nutrient availability) and column (cell lines) factors respectively. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Created in BioRender. https://BioRender.com/ am0jagq.

Article Snippet: Pdk1 shRNA Lentiviral particles (Cat. No. TL712182V, Origene) were mixed with 8 mg/ml of polybrene (Cat. No. TR-1003, Sigma-Aldrich) and added to cultured cells at a multiplicity of infection (MOI) of 500 or 120 (for the scrambled shRNA control).

Techniques: Activity Assay, Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy, Control, Knockdown, shRNA, Comparison

Journal: The Journal of biological chemistry

Article Title: Pyruvate dehydrogenase kinase 1 controls triacylglycerol hydrolysis in cardiomyocytes.

doi: 10.1016/j.jbc.2025.108398

Figure Lengend Snippet: Figure 4. Effect of PDK1 suppression on triacylglycerol metabolism. Cells were incubated in 375 mM of palmitic acid conjugated to BSA (6:1 M ratio) in complete culture medium for 18 h. A and B, fatty acid oxidation dependency and (C and D) capacity were measured from oxygen consumption rate measurements of 6 independent co-cultures spanning 3 experimental repeats. E, Triacylglycerol abundance was measured following BSA or palmitic acid incubation in complete culture medium, (F) nutrient deprived in 5.5 mM glucose, serum free culture media for 3 h. G, thin layer chromatograph of alkyne incorporated lipids in cells treated with 10 mM of alkyne palmitate for 18 h, then nutrient deprived in low glucose, serum-free media for 3 h (n = 3). Data are shown as mean ± SEM and were analyzed by a Two-way ANOVA followed by a post hoc Tukey multiple comparison test. For (B), ANOVA showed (F and p values) of (0.066 and 0.7995), (0.002534 and 0.9604), (18.12 and 0.0004) for interaction, row (PA treatment) and column (cell lines) factors respectively. For (D), ANOVA showed (F and p values) of (1.326 and 0.2631), (40.91 and < 0.0001), (13.16 and 0.0017) for interaction, row (PA treatment) and column (cell lines) factors respectively. For (E), ANOVA showed (F and p values) of (0.1336 and 0.7190), (30.66 and < 0.0001), (0.4757 and 0.4992) for interaction, row (PA treatment) and column (cell lines) factors respectively. For (F), ANOVA showed (F and p values) of (30.91 and 0.0001), (250.2 and < 0.0001), (27.78 and 0.0002) for interaction, row (nutrient availability) and column (cell lines) factors respectively. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Pdk1 shRNA Lentiviral particles (Cat. No. TL712182V, Origene) were mixed with 8 mg/ml of polybrene (Cat. No. TR-1003, Sigma-Aldrich) and added to cultured cells at a multiplicity of infection (MOI) of 500 or 120 (for the scrambled shRNA control).

Techniques: Incubation, Comparison

Journal: The Journal of biological chemistry

Article Title: Pyruvate dehydrogenase kinase 1 controls triacylglycerol hydrolysis in cardiomyocytes.

doi: 10.1016/j.jbc.2025.108398

Figure Lengend Snippet: Figure 5. Comparative analyses of lipid profiles. A, principal component analysis of lipid abundances in control and Pdk1 knockdown cells treated with 375 mM palmitic acid for 18 h and nutrient deprived in low glucose, serum-free media for 3 h. Relative lipid composition in (B) control and (C) Pdk1 knockdown cells following palmitic acid treatment. D, Volcano plot depicting lipid abundances in Pdk1 knockdown cells relative to control following palmitic acid treatment. Blue and red dots indicate downregulation and upregulation in Pdk1 knockdown cells respectively. E, normalized abundance of all identified triacylglycerol species in cells following palmitic acid treatment. Sum abundance of triacylglycerol species of certain (F) chain length and (G) unsaturation in cells following palmitic acid treatment. H, slope comparisons of lipid classes in control and Pdk1 knockdown cells following nutrient deprivation for 3 h. I, total triacylglycerol abundance during nutrient deprivation, with time = 0 representing the condition in the immediate aftermath of palmitic acid treatment without nutrient deprivation. Cells were treated with 150 mM [U-13C16] palmitate, then incubated in low glucose, serum-free media

Article Snippet: Pdk1 shRNA Lentiviral particles (Cat. No. TL712182V, Origene) were mixed with 8 mg/ml of polybrene (Cat. No. TR-1003, Sigma-Aldrich) and added to cultured cells at a multiplicity of infection (MOI) of 500 or 120 (for the scrambled shRNA control).

Techniques: Control, Knockdown, Incubation

Journal: The Journal of biological chemistry

Article Title: Pyruvate dehydrogenase kinase 1 controls triacylglycerol hydrolysis in cardiomyocytes.

doi: 10.1016/j.jbc.2025.108398

Figure Lengend Snippet: Figure 6. Subcellular mapping of carbohydrate and lipid metabolism proteins. Statistically significant (p < 0.05) differentially abundant metabolism- related proteins between control and Pdk1 knockdown cells treated with (A) 375 mM palmitic acid for 18 h, (B) then nutrient deprived in low glucose, serum- free media for 3 h, were connected in a high-confidence (functional score 0.80) protein-protein interaction network (purple lines) using STRING, and depicted in the context of their sub-cellular locations, derived from literature. The color of the nodes represents the fold change in Pdk1 knockdown cells relative to control, while the thickness of the line around the nodes represents the adjusted p-value. The shape of the node represents the metabolism pathway according to the Reactome database. Circular nodes represent involvement in lipid metabolism, square nodes represent involvement in carbo- hydrate metabolism, and diamond nodes represent involvement in both carbohydrate and lipid metabolism. And the shape of the node represents the metabolism pathway according to the Reactome.

Article Snippet: Pdk1 shRNA Lentiviral particles (Cat. No. TL712182V, Origene) were mixed with 8 mg/ml of polybrene (Cat. No. TR-1003, Sigma-Aldrich) and added to cultured cells at a multiplicity of infection (MOI) of 500 or 120 (for the scrambled shRNA control).

Techniques: Control, Knockdown, Functional Assay, Derivative Assay

Journal: The Journal of biological chemistry

Article Title: Pyruvate dehydrogenase kinase 1 controls triacylglycerol hydrolysis in cardiomyocytes.

doi: 10.1016/j.jbc.2025.108398

Figure Lengend Snippet: Figure 7. Predicted enzymatic activity. Algorithmic prediction of enzyme activity based on our lipidomics dataset was performed on the LipidOne platform (v 2.1). Activity prediction was carried out in Pdk1 knockdown cells relative to control in the (A) palmitic acid-treated condition and the (B) nutrient- deprived condition. The color of the nodes represents the activation status in Pdk1 knockdown cells relative to the control. Nodes in cream represent proteins not identified by the algorithm but shown nonetheless to provide increased biochemical context. The color of the line around the node represents the fold change of the protein abundance in Pdk1 knockdown cells relative to the control derived from our proteomics dataset. The thickness of the line around the node represents the adjusted p-value.

Article Snippet: Pdk1 shRNA Lentiviral particles (Cat. No. TL712182V, Origene) were mixed with 8 mg/ml of polybrene (Cat. No. TR-1003, Sigma-Aldrich) and added to cultured cells at a multiplicity of infection (MOI) of 500 or 120 (for the scrambled shRNA control).

Techniques: Activity Assay, Knockdown, Control, Activation Assay, Cream, Quantitative Proteomics, Derivative Assay

Journal: Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia

Article Title: Role for loss of nuclear PTEN in a harbinger of brain metastases.

doi: 10.1016/j.jocn.2017.06.004

Figure Lengend Snippet: Fig. 1. Examples of the PTEN and PDK-1 expression levels.

Article Snippet: Diluted mouse monoclonal (dilution 1:100) antibodies for PTEN (antibody PTEN; Dako, Glostrup, Denmark) and rabbit polyclonal (dilution 1:200) antibodies for PDK1 (antibody PDK1; OriGene Technologies, Inc., Rockville, USA) were employed as primary antibodies.

Techniques: Expressing

Journal: Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia

Article Title: Role for loss of nuclear PTEN in a harbinger of brain metastases.

doi: 10.1016/j.jocn.2017.06.004

Figure Lengend Snippet: Fig. 2a. Results of the PTEN and PDK-1 expression levels in all cancers.

Article Snippet: Diluted mouse monoclonal (dilution 1:100) antibodies for PTEN (antibody PTEN; Dako, Glostrup, Denmark) and rabbit polyclonal (dilution 1:200) antibodies for PDK1 (antibody PDK1; OriGene Technologies, Inc., Rockville, USA) were employed as primary antibodies.

Techniques: Expressing

Journal: Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia

Article Title: Role for loss of nuclear PTEN in a harbinger of brain metastases.

doi: 10.1016/j.jocn.2017.06.004

Figure Lengend Snippet: Fig. 2b. Results of the PTEN and PDK-1 expression levels in lung cancer.

Article Snippet: Diluted mouse monoclonal (dilution 1:100) antibodies for PTEN (antibody PTEN; Dako, Glostrup, Denmark) and rabbit polyclonal (dilution 1:200) antibodies for PDK1 (antibody PDK1; OriGene Technologies, Inc., Rockville, USA) were employed as primary antibodies.

Techniques: Expressing

Journal: Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia

Article Title: Role for loss of nuclear PTEN in a harbinger of brain metastases.

doi: 10.1016/j.jocn.2017.06.004

Figure Lengend Snippet: Fig. 2c. Results of the PTEN and PDK-1 expression levels in breast cancer.

Article Snippet: Diluted mouse monoclonal (dilution 1:100) antibodies for PTEN (antibody PTEN; Dako, Glostrup, Denmark) and rabbit polyclonal (dilution 1:200) antibodies for PDK1 (antibody PDK1; OriGene Technologies, Inc., Rockville, USA) were employed as primary antibodies.

Techniques: Expressing

Journal: Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia

Article Title: Role for loss of nuclear PTEN in a harbinger of brain metastases.

doi: 10.1016/j.jocn.2017.06.004

Figure Lengend Snippet: Fig. 2d. Results of the PTEN and PDK-1 expression levels in colorectal cancer.

Article Snippet: Diluted mouse monoclonal (dilution 1:100) antibodies for PTEN (antibody PTEN; Dako, Glostrup, Denmark) and rabbit polyclonal (dilution 1:200) antibodies for PDK1 (antibody PDK1; OriGene Technologies, Inc., Rockville, USA) were employed as primary antibodies.

Techniques: Expressing

Journal: Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia

Article Title: Role for loss of nuclear PTEN in a harbinger of brain metastases.

doi: 10.1016/j.jocn.2017.06.004

Figure Lengend Snippet: Fig. 2e. Results of the PTEN and PDK-1 expression levels in other cancers.

Article Snippet: Diluted mouse monoclonal (dilution 1:100) antibodies for PTEN (antibody PTEN; Dako, Glostrup, Denmark) and rabbit polyclonal (dilution 1:200) antibodies for PDK1 (antibody PDK1; OriGene Technologies, Inc., Rockville, USA) were employed as primary antibodies.

Techniques: Expressing

Journal: Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia

Article Title: Role for loss of nuclear PTEN in a harbinger of brain metastases.

doi: 10.1016/j.jocn.2017.06.004

Figure Lengend Snippet: Fig. 3. Examples of the PTEN and PDK-1 expression levels in lung cancer 72 years old female. Images of lung small cell carcinoma and left frontal lobe metastases and immunohistochemical staining of PTEN & PDK-1 (original magnification: 200) a. Thoracic CT image b. MRI, T1-weighted gadolinium-enhanced image c. origin; cytoplasmic PTEN: strong positive, nuclear PTEN: strong positive d. METs; cytoplasmic PTEN: positive, nuclear PTEN: negative e. origin; PDK-1: positive f. METs; PDK-1: strong positive.

Article Snippet: Diluted mouse monoclonal (dilution 1:100) antibodies for PTEN (antibody PTEN; Dako, Glostrup, Denmark) and rabbit polyclonal (dilution 1:200) antibodies for PDK1 (antibody PDK1; OriGene Technologies, Inc., Rockville, USA) were employed as primary antibodies.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: PDK1-Foxo1 in Agouti-Related Peptide Neurons Regulates Energy Homeostasis by Modulating Food Intake and Energy Expenditure

doi: 10.1371/journal.pone.0018324

Figure Lengend Snippet: ( A ) Representative immunofluorescence images of hypothalamic regions doubly labeled for AGRP and PDK1 in 24-week-old AGRPPdk1 +/+ (top panel) and AGRPPdk1 −/− (bottom panel) mice fed ad libitum . Green, red and blue indicate AGRP, PDK1, and DAPI staining, respectively. White arrows indicate AGRP neurons. Scale bars indicate 50 µm. ( B ) Quantification of PDK1-positive AGRP neurons from AGRPPdk1 +/+ (gray bar) and AGRPPdk1 −/− (blue bar) mice. The percentage of PDK1-positive AGRP neurons among AGRP neurons was determined as described in Experimental Procedures. Values are means ± SEM of three mice of each genotype. An asterisk indicates a statistically significant difference between AGRPPdk1 +/+ and AGRPPdk1 −/− mice with p<0.001 (one-factor ANOVA). ( C ) Body weight of male (left panel) and female (right panel) control (open circles) and AGRPPdk1 −/− (closed circles) mice fed normal chow. Values represent mean ± SEM of 35–40 mice in each genotype. An asterisk indicates a statistically significant difference between control and AGRPPdk1 −/− mice with p<0.05 (one-factor ANOVA). ( D ) Naso-anal length (NAL) of male (left panel) and female (right panel) control mice (gray bar) and AGRPPdk1 −/− mice (blue bar) at 24 weeks of age. Values represent mean ± SEM of 30 mice in each genotype. An asterisk indicates a statistically significant difference between control and AGRPPdk1 −/− mice with p<0.05 (one-factor ANOVA). ( E ) The calculated weights of muscle, adipose tissue, visceral fat, and subcutaneous fat in male control (gray bar) and AGRPPdk1 −/− (blue bar) mice at 24 weeks of age. Skeletal muscle weight, adipose tissue weight (sum of visceral and subcutaneous fats), visceral fat weight, and subcutaneous fat weight were calculated as described in Experimental Procedures. The data represent the mean weight ± SEM of 10 mice per genotype. Asterisks indicate statistically significant differences between control and AGRPPdk1 −/− mice with *p<0.001, **p<0.005, and ***p<0.05 (one-factor ANOVA). ( F ) Four-day food intake of male and female control mice (gray bar) and AGRPPdk1 −/− mice (blue bar) at 24 weeks of age. Values represent mean ± SEM of 40 mice in each genotype. An asterisk indicates a statistically significant difference between control and AGRPPdk1 −/− mice with p<0.05 (one-factor ANOVA). ( G ) Oxygen consumption in male control (gray bar) and AGRPPdk1 −/− (blue bar) mice at 24 weeks of age. The values represent the mean ± SEM of 10 male mice per genotype. ( H ) Respiratory quotient of male control (gray bar) and AGRPPdk1 −/− (blue bar) mice at 24 weeks of age. The values represent the mean ± SEM of 10 mice per genotype. ( I ) Basal locomotor activity for male control (gray bar) and AGRPPdk1 −/− (blue bar) mice at 24 weeks of age during the light and dark phases. The values represent the mean ± SEM of 5 mice per genotype. ( J–K ) Effects of intraperitoneal leptin injections on body weight and food intake. Leptin was injected into control (open circle, n = 8) or AGRPPdk1 −/− mice (closed circle, n = 5) for three consecutive days. Body weight (J) and food intake (K) were monitored for seven days. Body weight and food intake are presented as percentages of values from saline-injected mice for each genotype. An asterisk in (J) indicates a statistically significant difference between control and AGRPPdk1 −/− mice with p<0.05 (one-factor ANOVA). An asterisk in (K) indicates a statistically significant difference between saline- and leptin-injected mice in AGRPPdk1 −/− mice with p<0.05 (one-factor ANOVA).

Article Snippet: The primary antibodies were Ab-241 (Signalway Antibody, Pearland, TX) for PDK1 and GT15023 (Neuromics, Edina, MN) for AGRP; the secondary antibodies were Alexa Fluor R 594 chicken anti-rabbit IgG and Alexa Fluor R 488 donkey anti-goat IgG (Molecular Probes, Eugene, OR).

Techniques: Immunofluorescence, Labeling, Staining, Control, Activity Assay, Injection, Saline

Journal: PLoS ONE

Article Title: PDK1-Foxo1 in Agouti-Related Peptide Neurons Regulates Energy Homeostasis by Modulating Food Intake and Energy Expenditure

doi: 10.1371/journal.pone.0018324

Figure Lengend Snippet: ( A ) Subcellular localization of Foxo1 in AGRP neurons from AGRPPdk1 +/+ (top panel) and AGRPPdk1 −/− (bottom panel) mice. Representative double immunofluorescence images show hypothalamic regions doubly-labeled for AGRP and Foxo1. Green, AGRP; red, Foxo1; blue, DAPI staining. White arrows indicate AGRP neurons. ( B ) Quantification of nuclear Foxo1 in AGRP neurons from AGRPPdk1 +/+ and AGRPPdk1 −/− mice. The percentage of AGRP neurons with nuclear Foxo1 among all AGRP neurons was determined as described in Experimental Procedures. Values are means ± SEM of three mice in each genotype. An asterisk indicates a statistically significant difference between AGRPPdk1 +/+ and AGRPPdk1 −/− mice with p<0.001 (one-factor ANOVA). ( C ) Body weights of male control (open circles), Δ256Foxo1 AGRP (open triangles), AGRPPdk1 −/− (closed circles), and Δ256Foxo1 AGRP Pdk1 −/− (closed triangles) mice. Values represent the mean ± SEM of 20 mice in each genotype. Asterisks indicate statistically significant differences between AGRPPdk1 −/− and Δ256Foxo1 AGRP Pdk1 −/− with *p<0.001, **p<0.01, and ***p<0.05 (one-factor ANOVA). ( D ) The calculated weights of muscle, adipose tissue, visceral fat, and subcutaneous fat in male control (gray bar), Δ256Foxo1 AGRP (white bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice at 24 weeks of age. Skeletal muscle weight, adipose tissue weight (sum of visceral and subcutaneous fats), visceral fat weight, and subcutaneous fat weight were calculated as described in Experimental Procedures. The data represent the mean weight ± SEM of 10 mice per genotype. Asterisks indicate statistically significant differences as indicated with *p<0.001, **p<0.005, and ***p<0.05 (one-factor ANOVA). ( E ) Four-day food intake for male control (gray bar), Δ256Foxo1 AGRP (white bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice at 24 weeks of age. The data represent the mean ± SEM of 10 mice per genotype. An asterisk indicates a statistically significant difference as indicated with *p<0.005 (one-factor ANOVA). ( F ) Expression in the fed-state of hypothalamic neuropeptide genes in control (gray bar), Δ256Foxo1 AGRP (white bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice. Data were normalized to β-actin expression and represent the mean ± SEM of six mice per genotype. An asterisk indicates a statistically significant difference between control and Δ256Foxo1 AGRP Pdk1 −/− mice with p<0.05 (one-factor ANOVA). ( G ) Oxygen consumption in male control (gray bar), Δ256Foxo1 AGRP (white bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice at 24 weeks of age. The values represent the mean ± SEM of 10 male mice per genotype. ( H ) Respiratory quotient of male control (gray bar), Δ256Foxo1 AGRP (white bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice at 24 weeks of age. The values represent the mean ± SEM of 10 mice per genotype. An asterisk indicates a statistically significant difference between AGRPPdk1 −/− and Δ256Foxo1 AGRP Pdk1 −/− mice with p<0.05 (one-factor ANOVA). ( I ) Basal locomotor activity for male control (gray bar), Δ256Foxo1 AGRP (white bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice at 24 weeks of age during the light and dark phases. The values represent the mean ± SEM of 5 mice per genotype. An asterisk indicates a statistically significant difference as indicated with p<0.001 (one-factor ANOVA).

Article Snippet: The primary antibodies were Ab-241 (Signalway Antibody, Pearland, TX) for PDK1 and GT15023 (Neuromics, Edina, MN) for AGRP; the secondary antibodies were Alexa Fluor R 594 chicken anti-rabbit IgG and Alexa Fluor R 488 donkey anti-goat IgG (Molecular Probes, Eugene, OR).

Techniques: Immunofluorescence, Labeling, Staining, Control, Expressing, Activity Assay

Journal: PLoS ONE

Article Title: PDK1-Foxo1 in Agouti-Related Peptide Neurons Regulates Energy Homeostasis by Modulating Food Intake and Energy Expenditure

doi: 10.1371/journal.pone.0018324

Figure Lengend Snippet: ( A ) Effects of 4 weeks of calorie restriction on body weight of 20-week-old male control (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice. Values represent means ± SEM of the percentages of reduction of body weight at the end (4 week) of pair feeding compared to at the beginning of pair feeding. An asterisk indicates a statistically significant difference between bodyweight at the beginning and at the end of calorie restriction with p<0.05 (one-factor ANOVA; n = 10 male mice per genotype). ( B ) Oxygen consumption of male calorie-restricted control (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) during the light and dark phases. Values represent the mean ± SEM of 10 mice per genotype. An asterisk indicates a statistically significant difference between AGRPPdk1 −/− and control or Δ256Foxo1 AGRP Pdk1 −/− mice with p<0.05 (one-factor ANOVA). ( C ) Respiratory quotients of male pair-fed control (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice during the light and dark phases. Values represent the mean ± SEM of 10 mice per genotype. An asterisk indicates a statistically significant difference between AGRPPdk1 −/− and control or Δ256Foxo1 AGRP Pdk1 −/− mice with p<0.001 (one-factor ANOVA). ( D ) Basal locomotor activity of male pair-fed control (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) during the light and dark phases. Values represent the mean ± SEM of 10 mice per genotype. An asterisk indicates a statistically significant difference between AGRPPdk1 −/− and Δ256Foxo1 AGRP Pdk1 −/− mice with p<0.05 (one-factor ANOVA). ( E–F ) Effects of intraperitoneal leptin injections on body weight and food intake of pair-fed control (open squares, n = 10), AGRPPdk1 −/− mice (blue closed circles, n = 10), and Δ256Foxo1 AGRP Pdk1 −/− (red squares, n = 10) mice. Leptin was injected on 3 consecutive days. Body weight (E) and food intake (F) were monitored for eight days. Body weight and food intake data are presented as the percentage of that for saline-injected mice for each genotype. An asterisk in (F) indicates statistically significant differences between control and AGRPPdk1 −/− mice with *p<0.001, **p<0.01, and ***p<0.05 by one-factor ANOVA. ( G ) Cumulative food intake for male pair-fed control (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice injected with saline or ghrelin after a 24-h fast. Cumulative food intake was monitored for 6 h. Values represent the ratio of the mean cumulative food intakes of ghrelin:vehicle injected mice for each genotype. An asterisk indicates statistically significant differences between control and AGRPPdk1 −/− mice with *p<0.02 (one-factor ANOVA; n = 10 per genotype for each condition). ( H ) Cumulative food intake of male pair-fed control (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice injected with saline or MTII after a 24-h fast. Cumulative food intake was monitored for 24 h. Values represent the ratio of the mean cumulative food intakes of MTII:vehicle injected mice for each genotype. Asterisks indicate statistically significant differences between control and AGRPPdk1 −/− mice with p<0.05 (one-factor ANOVA; n = 10 per genotype).

Article Snippet: The primary antibodies were Ab-241 (Signalway Antibody, Pearland, TX) for PDK1 and GT15023 (Neuromics, Edina, MN) for AGRP; the secondary antibodies were Alexa Fluor R 594 chicken anti-rabbit IgG and Alexa Fluor R 488 donkey anti-goat IgG (Molecular Probes, Eugene, OR).

Techniques: Control, Activity Assay, Injection, Saline

Journal: PLoS ONE

Article Title: PDK1-Foxo1 in Agouti-Related Peptide Neurons Regulates Energy Homeostasis by Modulating Food Intake and Energy Expenditure

doi: 10.1371/journal.pone.0018324

Figure Lengend Snippet: ( A )–( C ) Administration of 10 −10 M of ghrelin increased cytosolic calcium ([Ca 2+ ] i ) in AGRP neurons from AGRPPdk1 +/+ (A), AGRPPdk1 −/− (B), and Δ256Foxo1 AGRP Pdk1 −/− (C) mice. Administration of 10 −12 M of leptin inhibited increases in [Ca 2+ ] i induced by ghrelin in AGRPPdk1 +/+ (A) and Δ256Foxo1 AGRP Pdk1 −/− (B) neurons, but not in AGRPPdk1 −/− neurons (C). ( D ) Amplitude of [Ca 2+ ] i responses to ghrelin in the absence or presence of leptin (10 −12 M) in AGRP neurons of AGRPPdk1 +/+ (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice. Values are means ± SEM of the ratio of [Ca 2+ ] i in the presence and absence of leptin. An asterisk indicates a statistically significant difference between the absence and the presence of leptin with p<0.05 (one-factor ANOVA: n = 10 AGRP neurons per genotype). ( E ) Fura-2 detection of [Ca 2+ ] i in response to ghrelin. Values are means ± SEM of ghrelin-stimulated fura-2 ratios in AGRP neurons from AGRPPdk1 +/+ (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar). An asterisk indicates a statistically significant difference between AGRPPdk1 −/− and control or Δ256Foxo1 AGRP Pdk1 −/− mice with p<0.05 (one-factor ANOVA: n = 10 AGRP neurons per genotype).

Article Snippet: The primary antibodies were Ab-241 (Signalway Antibody, Pearland, TX) for PDK1 and GT15023 (Neuromics, Edina, MN) for AGRP; the secondary antibodies were Alexa Fluor R 594 chicken anti-rabbit IgG and Alexa Fluor R 488 donkey anti-goat IgG (Molecular Probes, Eugene, OR).

Techniques: Control

Journal: bioRxiv

Article Title: Cardiac REDD1 alters glucose and fatty acid metabolic gene expression via an mTORC1-independent, PPARα-dependent mechanism and drives hypertrophic growth

doi: 10.64898/2026.03.16.710895

Figure Lengend Snippet: A-D. Hearts of adult (8-14-week-old) male and female mice of the indicated genotypes were harvested and subjected to total RNA extraction and qPCR for the indicated genes. (A-B) n=9,11 ( Redd1) , n=12,12 ( Pdk1, Pdk2, Pdp1 ), n=10,12 ( Pdk3 ), and n=11,12 ( Pdk4, Pdp2 ), unpaired t test, 2-way ANOVA. (C-D) n=3,3,3,4 ( Redd1, Pdk1, Pdk2, Pdk3, Pdp1, Pdp2 ) and n=3,3,3,3 ( Pdk4 ), 1-way ANOVA, 2-way ANOVA. E-J. Hearts of adult (8-14-week-old) male and female mice of the indicated genotypes were harvested, lysed, and subjected to western blotting with the indicated antibodies. Signals were quantified with densitometry, normalized to total PDH, and plotted. (E-G) n=10,19 (pPDH (Ser293), pPDH (Ser300)), unpaired t test. (H-J) n=5,6 (pPDH (Ser293), pPDH (Ser300)), unpaired t test. Error bars represent SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. M = marker.

Article Snippet: 2 μg RNA was reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was performed using TaqMan Gene Expression Assays (ThermoFisher Scientific) and the QuantStudio 3 Real-Time PCR System (ThermoFisher Scientific) for the following genes: 18S (Mm03928990_g1), Redd1 (Mm00512504_g1), PDK1 (Hs05380290_s1), Pdk1 (Rn00587598_m1), PDK2 (Hs04965351_m1), Pdk2 (Rn00446679_m1), PDK3 (Hs03878443_s1), Pdk3 (Rn01424337_m1), PDK4 (Hs01037712_m1), Pdk4 (Rn00585577_m1), PDP1 (Hs01081518_s1), Pdp1 (Rn01437077_m1), PDP2 (Hs01934174_s1), Pdp2 (Mm02526496_s1), ACSL1 (Hs00960561_m1), or Nppb (Mm01255770_g1).

Techniques: RNA Extraction, Western Blot, Marker