Journal: Scientific Reports

Article Title: Functional Consequences of PDK4 Deficiency in Doberman Pinscher Fibroblasts

doi: 10.1038/s41598-020-60879-6

Figure Lengend Snippet: PDK isoform transcription levels in response to starvation. Relative transcription levels of ( A ) PDK1 , ( B ) PDK2 , ( C ) PDK3 , and ( D ) PDK4 in cells representing each of the three groups PDK4 wt/wt , PDK4 wt/del , and PDK4 del/del cells in unstarved and starved conditions. (Data presented as mean + std. err. *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: PDK2 , Thermo Scientific #Cf02700630_g1.

Techniques:

Journal: Scientific Reports

Article Title: Functional Consequences of PDK4 Deficiency in Doberman Pinscher Fibroblasts

doi: 10.1038/s41598-020-60879-6

Figure Lengend Snippet: Designed primers based on previous studies used for quantitative real-time PCR.

Article Snippet: PDK2 , Thermo Scientific #Cf02700630_g1.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Suppression of Pyruvate Dehydrogenase Kinase by Dichloroacetate in Cancer and Skeletal Muscle Cells Is Isoform Specific and Partially Independent of HIF-1α

doi: 10.3390/ijms22168610

Figure Lengend Snippet: DCA reduces the abundance of PDK1 in MDA-MB-231 and PC-3 cells. ( A , B ) Cancer cells were treated with 10 mM DCA and/or 5 mM metformin (MF) in serum-free RPMI medium for 24 h. The mRNA expression of PDK1–4 isoenzymes was evaluated in ( A ) MDA-MB-231 and ( B ) PC-3 cells with qPCR (endogenous control: cyclophilin). ( C – F ) After treating MDA-MB-231 and PC-3 cells with the PDK1 siRNA (siPDK1) or the scrambled siRNA (siSCR), cells were exposed to 10 mM DCA for 24 h in serum-free RPMI medium. Immunoblotting was used to evaluate the protein levels of ( C ) PDK1, ( D ) PDK2, ( E ) PDK3, and ( F ) phospho-PDHE1α Ser293 (pPDHE1α). ( G ) Ponceau S staining was used to evaluate the total protein loading. Numbers next to the blots and Ponceau stains indicate molecular weight markers in kDa. ( H ) Lactate production in PDK1-deficient (siPDK1) and control (siSCR) cells treated with 5 mM metformin (MF) for 24 h. Results are means ± SD of one experiment performed in triplicate for each cell line ( n = 3). * p < 0.05 vs. untreated cells ( A , B ) or siSCR ( C – F , H ) and # p < 0.05 as indicated.

Article Snippet: MEMα with nucleosides (MEMα+), MEMα without nucleosides (MEMα−), fetal bovine serum (FBS), Pen Strep (5000 U/mL of penicillin and 5000 μg/mL of streptomycin), Fungizone (250 μg/mL of amphotericin B), Pierce BCA Protein Assay Kit, Pierce Enhanced Chemiluminescence (ECL) Western Blotting Substrate, PDK1 siRNA (4427038), scrambled siRNA (AM4611), transfection reagent Lipofectamine 2000, High-Capacity cDNA Reverse Transcription Kit, MicroAmp optical 96-well reaction plates, MicroAmp optical adhesive films, TaqMan Universal Master Mix and TaqMan gene expression assays for human HIF-1α (Hs00153153_m1), HIF-1β (Hs00231048_m1), PGK1 (4333765F), PDK1 (Hs01561847_m1), PDK2 (Hs00176865_m1), PDK3 (Hs00178440_m1), PDK4 (Hs01037712_m1), CLPP (Hs01548165_m1), AFG3L2 (Hs01064997_m1), VEGF (Hs00900054_m1), cyclophilin (PPIA) (Hs99999904_m1) and for rat PDK1 (Rn00587598_m1), PDK2 (Rn00446679_m1), PDK3 (Rn01424337_m1), PDK4 (Rn00585577_m1), and β-actin (Rn00667869_m1) were from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Expressing, Control, Western Blot, Staining, Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: Suppression of Pyruvate Dehydrogenase Kinase by Dichloroacetate in Cancer and Skeletal Muscle Cells Is Isoform Specific and Partially Independent of HIF-1α

doi: 10.3390/ijms22168610

Figure Lengend Snippet: DCA reduces the abundance of PDK1 and PDK2 in L6 myotubes despite upregulation of HIF-1α and inhibition of the proteasome. ( A – H ) L6 myotubes were treated with 10 mM DCA or vehicle in serum-free minimal essential medium-α (MEMα). After 12 h of treatment, medium was removed, cells were washed with PBS and fresh MEMα− with vehicle (V), 10 mM DCA (D), and/or 0.5 μg/mL puromycin (P) was added for the next 12 h. ( B ) The gene expression levels of PDK1, PDK2, PDK3, and PDK4 were analyzed with qPCR (endogenous control: β-actin). Immunoblotting was used to estimate the protein abundance of ( C ) PDK1, PDK2, and PDK3, ( D ) phospho-PDHE1α Ser293 (pPDHE1α), ( E ) HIF-1α, ( F ) phospho-AMPK Thr172 (pAMPK), and ( G ) phospho-ACC Ser79 (pACC). ( H ) A representative Ponceau S staining of the membrane. Results are means ± SD of two independent experiments, each performed in three replicates ( n = 6). * p < 0.05 vs. V/V and # p ≤ 0.05 as indicated. ( I – M ) The differentiated L6 cells (myotubes) were treated with MG-132 (1 or 10 μM MG) for 1 h, which was followed by a 24-h treatment with DCA (10 mM) and/or MG-132 (1 or 10 μM). Immunoblotting was used to estimate the abundance of ( I ) HIF-1α, ( J ) PDK1, ( K ) PDK2, and ( L ) PDK3. ( M ) Protein loading was evaluated with the Ponceau S staining. Numbers next to the blots and Ponceau stains indicate molecular weight markers in kDa. Results are means ± SD of one experiment in four replicates ( n = 4). * p < 0.05 vs. Control and # p < 0.05 as indicated.

Article Snippet: MEMα with nucleosides (MEMα+), MEMα without nucleosides (MEMα−), fetal bovine serum (FBS), Pen Strep (5000 U/mL of penicillin and 5000 μg/mL of streptomycin), Fungizone (250 μg/mL of amphotericin B), Pierce BCA Protein Assay Kit, Pierce Enhanced Chemiluminescence (ECL) Western Blotting Substrate, PDK1 siRNA (4427038), scrambled siRNA (AM4611), transfection reagent Lipofectamine 2000, High-Capacity cDNA Reverse Transcription Kit, MicroAmp optical 96-well reaction plates, MicroAmp optical adhesive films, TaqMan Universal Master Mix and TaqMan gene expression assays for human HIF-1α (Hs00153153_m1), HIF-1β (Hs00231048_m1), PGK1 (4333765F), PDK1 (Hs01561847_m1), PDK2 (Hs00176865_m1), PDK3 (Hs00178440_m1), PDK4 (Hs01037712_m1), CLPP (Hs01548165_m1), AFG3L2 (Hs01064997_m1), VEGF (Hs00900054_m1), cyclophilin (PPIA) (Hs99999904_m1) and for rat PDK1 (Rn00587598_m1), PDK2 (Rn00446679_m1), PDK3 (Rn01424337_m1), PDK4 (Rn00585577_m1), and β-actin (Rn00667869_m1) were from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Inhibition, Gene Expression, Control, Western Blot, Quantitative Proteomics, Staining, Membrane, Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: Suppression of Pyruvate Dehydrogenase Kinase by Dichloroacetate in Cancer and Skeletal Muscle Cells Is Isoform Specific and Partially Independent of HIF-1α

doi: 10.3390/ijms22168610

Figure Lengend Snippet: Overview of the antibodies used for immunoblotting. Abbreviations: Ab—antibody, Mo—mouse, O/N—overnight, Rb—rabbit. Cell Signaling Technology (Danvers, MA, USA), Novus Biologicals (Centennial, CO, USA), Abcam (Cambridge, UK).

Article Snippet: MEMα with nucleosides (MEMα+), MEMα without nucleosides (MEMα−), fetal bovine serum (FBS), Pen Strep (5000 U/mL of penicillin and 5000 μg/mL of streptomycin), Fungizone (250 μg/mL of amphotericin B), Pierce BCA Protein Assay Kit, Pierce Enhanced Chemiluminescence (ECL) Western Blotting Substrate, PDK1 siRNA (4427038), scrambled siRNA (AM4611), transfection reagent Lipofectamine 2000, High-Capacity cDNA Reverse Transcription Kit, MicroAmp optical 96-well reaction plates, MicroAmp optical adhesive films, TaqMan Universal Master Mix and TaqMan gene expression assays for human HIF-1α (Hs00153153_m1), HIF-1β (Hs00231048_m1), PGK1 (4333765F), PDK1 (Hs01561847_m1), PDK2 (Hs00176865_m1), PDK3 (Hs00178440_m1), PDK4 (Hs01037712_m1), CLPP (Hs01548165_m1), AFG3L2 (Hs01064997_m1), VEGF (Hs00900054_m1), cyclophilin (PPIA) (Hs99999904_m1) and for rat PDK1 (Rn00587598_m1), PDK2 (Rn00446679_m1), PDK3 (Rn01424337_m1), PDK4 (Rn00585577_m1), and β-actin (Rn00667869_m1) were from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Suppression of Pyruvate Dehydrogenase Kinase by Dichloroacetate in Cancer and Skeletal Muscle Cells Is Isoform Specific and Partially Independent of HIF-1α

doi: 10.3390/ijms22168610

Figure Lengend Snippet: Overview of the antibodies used for immunoblotting. Abbreviations: Ab—antibody, Mo—mouse, O/N—overnight, Rb—rabbit. Cell Signaling Technology (Danvers, MA, USA), Novus Biologicals (Centennial, CO, USA), Abcam (Cambridge, UK).

Article Snippet: PDK2 , 46 , Novus Biologicals , NBP1-87307 , Rb , 1:800 , O/N , Bio-Rad , 1706515 , 1:8000 , 1 h.

Techniques: Western Blot