primary antibodies against akt, phospho-akt, pdk1, phosphopdk1, pi3k, phospho-pi3k  (Cell Signaling Technology Inc)

Journal: Cancer & Metabolism

Article Title: PDK1- and PDK2-mediated metabolic reprogramming contributes to the TGFβ1-promoted stem-like properties in head and neck cancer

doi: 10.1186/s40170-022-00300-0

Figure Lengend Snippet: Expression levels of PDK2 were increased in higher tumor stages of HNC patients’ tissues. A Immunohistochemical staining was performed using anti-PDK1 on tissue microarray containing 70 cases. PDK1 levels were analyzed in various stages and grades of HNC. B Immunohistochemical staining was performed using anti-PDK2 on tissue microarray containing 70 cases. PDK2 levels were analyzed in various stages and grades of HNC. * p < 0.05

Article Snippet: Primary IHC antibodies against PDK1 (1:100) and PDK2 (1:600) were obtained from Santa Cruz (Santa Cruz Biotech.

Techniques: Expressing, Immunohistochemical staining, Staining, Microarray

Journal: Cancer & Metabolism

Article Title: PDK1- and PDK2-mediated metabolic reprogramming contributes to the TGFβ1-promoted stem-like properties in head and neck cancer

doi: 10.1186/s40170-022-00300-0

Figure Lengend Snippet: PDK1 and PDK2 knockdown were confirmed in HNC cells. A SAS and FaDu shLuc, shPDK1, and shPDK2 cells (1×10 6 cells/3 ml) were seeded in a 6-cm dish overnight. PDK1 and PDK2 were detected by RT-qPCR. B SAS shLuc, shPDK1, and shPDK2 cells (4×10 5 cells/3 ml) were seeded in a 6-cm dish for 48 h. The expression levels of PDK1 (left panel) and PDK2 (right panel) protein were detected by western blotting. C SAS shLuc, shPDK1#261, shPDK2#315 cells, FaDu shLuc, shPDK1#263, shPDK2#315 (4×10 5 cells/3 ml) were seeded in a 6-cm culture dish for 48 h. We tested the PDH activity of the cells (1×10 6 cells). * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0001

Article Snippet: Primary IHC antibodies against PDK1 (1:100) and PDK2 (1:600) were obtained from Santa Cruz (Santa Cruz Biotech.

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Western Blot, Activity Assay

Journal: Cancer & Metabolism

Article Title: PDK1- and PDK2-mediated metabolic reprogramming contributes to the TGFβ1-promoted stem-like properties in head and neck cancer

doi: 10.1186/s40170-022-00300-0

Figure Lengend Snippet: PDK1 and PDK2 silencing modulated TGF-β1-promoted Warburg effects in HNC cells. A SAS shLuc, shPDK1#261, and shPDK2#315 cells (4×10 5 cells/3 ml) were pretreated with or without 5 ng/ml TGF-β1 medium for 48 h. Cells (1×10 4 cells/200 μl) were then seeded in a 96-well plate with or without TGF-β1 for 24 h, and then the medium was changed to a serum-free medium. After incubation for 24 h, lactate concentration in the supernatant was examined using the L-Lactate Assay kit. B SAS shLuc, shPDK1#261, and shPDK2#315 cells and FaDu shLuc, shPDK1#263, and shPDK2#315 (4×10 5 cells/3 ml) treated with or without TGF-β1 were seeded in a 6-cm culture dish for 48 h. We measured the ATP production of the cells (4×10 5 cells). C SAS shLuc, shPDK1, and shPDK2 cells (1×10 6 cells/3 ml) were seeded in a 6-cm dish overnight. Metabolism gene expressions were detected by RT-qPCR. D SAS shLuc, shPDK1#261, and shPDK2#315 cells (1×10 6 cells/3 ml) treated with or without TGF-β1 were seeded in a 6-cm dish overnight. Metabolism gene expressions were detected by RT-qPCR. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0001

Article Snippet: Primary IHC antibodies against PDK1 (1:100) and PDK2 (1:600) were obtained from Santa Cruz (Santa Cruz Biotech.

Techniques: Incubation, Concentration Assay, Lactate Assay, Quantitative RT-PCR

Journal: Cancer & Metabolism

Article Title: PDK1- and PDK2-mediated metabolic reprogramming contributes to the TGFβ1-promoted stem-like properties in head and neck cancer

doi: 10.1186/s40170-022-00300-0

Figure Lengend Snippet: PDK1 and PDK2 knockdown decreased TGF-β1-promoted migration and invasion ability of HNC cells. A Transwell migration activities of SAS shLuc, shPDK1#261, and shPDK2#315 cells (2.5×10 4 /100 μl serum-free DMEM) and FaDu shLuc, shPDK1#263, and shPDK2#315 cells (3×10 4 /100 μl serum-free DMEM) were determined after 30 h. B Transwell invasion activities of SAS shLuc, shPDK1#261, and shPDK2#315 cells (3×10 4 cells/500 μl serum-free DMEM) and FaDu shLuc, shPDK1#263, and shPDK2#315 cells (4×10 4 cells/500 μl serum-free DMEM) were determined after 48 h. C SAS shLuc, shPDK1#261, and shPDK2#315 cells (4×10 5 /3 ml) were pretreated with 5 ng/ml TGF-β1 medium for 48 h. Migration activities of these cells were determined as described in A . D SAS and FaDu cells (4×10 5 /3 ml) were pretreated with TGF-β1 medium for 48 h. Invasion activities of these cells were determined as described in B . * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0001

Article Snippet: Primary IHC antibodies against PDK1 (1:100) and PDK2 (1:600) were obtained from Santa Cruz (Santa Cruz Biotech.

Techniques: Knockdown, Migration

Journal: Cancer & Metabolism

Article Title: PDK1- and PDK2-mediated metabolic reprogramming contributes to the TGFβ1-promoted stem-like properties in head and neck cancer

doi: 10.1186/s40170-022-00300-0

Figure Lengend Snippet: PDK1 and PDK2 silencing downregulated sphere formation ability, stemness gene, and multidrug resistance gene expression in HNC cells. A SAS and FaDu cells (2.5×10 2 cells/100 μl) were seeded in 96-well ultra-low attachment plates in sphere medium. Sphere numbers were counted after 10-day incubation. B SAS shLuc parental cells (1×10 6 cells/3 ml) were seeded in a 6-cm dish with sphere medium overnight. SAS cells (1×10 4 cells/2 ml) were seeded in 6-well ultra-low attachment plates in sphere medium for 12 days. Expression of stemness genes and multidrug resistance genes (MDR) were detected in parental cells (P) and sphere cells (1S) by RT-qPCR. C SAS shLuc, shPDK1#261, and shPDK2#315 cells (2.5×10 2 cells/100 μl) were seeded in 96-well ultra-low attachment plates in sphere medium with or without 10 ng/ml TGF-β1. Sphere numbers were counted after 10-day incubation. shLuc (1S) compared with shLuc (P): * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0001. shPDK (1S) compared with shLuc (1S): # p < 0.05; ## p < 0.01; ### p < 0.005; #### p < 0.0001

Article Snippet: Primary IHC antibodies against PDK1 (1:100) and PDK2 (1:600) were obtained from Santa Cruz (Santa Cruz Biotech.

Techniques: Gene Expression, Incubation, Expressing, Quantitative RT-PCR

Journal: Cancer & Metabolism

Article Title: PDK1- and PDK2-mediated metabolic reprogramming contributes to the TGFβ1-promoted stem-like properties in head and neck cancer

doi: 10.1186/s40170-022-00300-0

Figure Lengend Snippet: PDK1 and PDK2 knockdown inhibited migration ability and reversed cisplatin and gemcitabine resistance in SAS spheroids cells. A SAS shLuc, shPDK1#261, and shPDK2#315 cells (1×10 4 cells/2 ml) were seeded in 6-well ultra-low attachment plates in sphere medium with or without 10 ng/ml TGF-β1 sphere medium and cultured for 12 days. Migration activities of the spheroid cells (2.5×10 4 cells/100 μl 0.5% FBS DMEM) were determined after 30 h. Five fields were counted per filter in each group. B–D Parental (P) or spheroid (1S) cells of SAS shLuc, shPDK1#261, and shPDK2#315 cells (2×10 3 cells/100 μl) were seeded in 96-well white plates with different chemo drugs. Celltiter-Glo Luminescent Cell Viability Assay was accessed after 48-h incubation. shLuc (1S) compared with shLuc (P): * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0001. shPDK (1S) compared with shLuc (1S): # p < 0.05; ## p < 0.01

Article Snippet: Primary IHC antibodies against PDK1 (1:100) and PDK2 (1:600) were obtained from Santa Cruz (Santa Cruz Biotech.

Techniques: Knockdown, Migration, Cell Culture, Cell Viability Assay, Incubation

Journal: Cancer & Metabolism

Article Title: PDK1- and PDK2-mediated metabolic reprogramming contributes to the TGFβ1-promoted stem-like properties in head and neck cancer

doi: 10.1186/s40170-022-00300-0

Figure Lengend Snippet: Associations of PDK1 and PDK2 expression with clinical features of HNC patients

Article Snippet: Primary IHC antibodies against PDK1 (1:100) and PDK2 (1:600) were obtained from Santa Cruz (Santa Cruz Biotech.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Mitochondrial Sirtuin 4 Resolves Immune Tolerance in Monocytes by Rebalancing Glycolysis and Glucose Oxidation Homeostasis

doi: 10.3389/fimmu.2018.00419

Figure Lengend Snippet: Depleting sirtuin 4 (SIRT4) hampers recovery of PDC inhibition by increasing PDK1 expression during physiological resolution of acute inflammatory response. Control and SIRT4 knockdown or control and SIRT4 knockout THP-1 cells were stimulated with 1 µg/ml lipopolysaccharide (LPS). (A) PDC decarboxylase activity was evaluated by commercial colorimetric assay kit ( N = 3). (B) PDK1 mRNA transcription was measured using real-time RT-PCR. PDK1 protein levels (C) and PDC protein levels and S232 phosphorylated PDC E1α levels (D) were shown by immunoblotting. Bar graphs depict mean values ± SE of N = 3. PDC, pyruvate dehydrogenase complex, PDK1, pyruvate dehydrogenase kinase 1, GAPDH, glyceraldehyde 3-phosphate dehydrogenase, KD, knockdown, KO, knockout.

Article Snippet: Blots were blocked with 5% milk-PBST for 1 h at room temperature and probed for overnight at 4°C using primary antibodies against SIRT4, PDK1, PDC, p-PDC E1α-S 232 (Genetex), and GAPDH.

Techniques: Inhibition, Expressing, Control, Knockdown, Knock-Out, Activity Assay, Colorimetric Assay, Quantitative RT-PCR, Western Blot