Journal: Cells

Article Title: Role of Mitochondrial Glycerol-3-Phosphate Dehydrogenase in Metabolic Adaptations of Prostate Cancer

doi: 10.3390/cells9081764

Figure Lengend Snippet: Energy metabolism and mGPDH content in prostate cancer cells. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) ( A ), as well as OCR/ECAR ratio ( B ) in prostate cancer cells (LNCaP) compared to control epithelial cell line (PNT1A). Values were calculated from the rates in basal conditions, i.e. at the presence of 10 mM glucose, and determined by a Seahorse XFe analyzer ( n = 5). ( C ) Enzyme activity of mGPDH measured spectrophotometrically using 10 mM glycerol-3-phosphate as a substrate ( n = 6). ( D ) ROS generation in intact LNCaP cells compared to control PNT1A measured by the CM-H 2 DCFDA probe. To determine the FCCP-sensitive portion of ROS production, 1 μM uncoupler was used. ( E ) Cell lysates (15 μg protein) were separated on SDS-PAGE and mGPDH content was analyzed by Western blotting using a specific antibody against mGPDH, actin was used as a loading control. Representative blot of 5 independent experiments is depicted. Antibody signals were quantified densitometrically as the total mGPDH levels normalized to actin levels and the results are expressed as % of control values. ( F ) Processing of mGPDH was determined densitometrically as a ratio of the lower band and total mGPDH content ( n = 5). Data represent the means ± S.D., * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The normal human immortalized prostate epithelial cell line PNT1A (95012614, Merck/Sigma, Darmstadt, Germany) and androgen-sensitive human prostate cancer cell line LNCaP (kindly provided by Dr. Hodny, IMG CAS, Prague, Czech Republic) and human embryonic kidney cells (HEK293) were purchased from ATCC (CRL-1573, ATCC, Manassas, VA, USA).

Techniques: Activity Assay, SDS Page, Western Blot

Journal: Cells

Article Title: Role of Mitochondrial Glycerol-3-Phosphate Dehydrogenase in Metabolic Adaptations of Prostate Cancer

doi: 10.3390/cells9081764

Figure Lengend Snippet: Analysis of mGPDH forms. ( A ) mGPDH topology by the Protter visualization tool. Predicted phosphorylation (green) and targeting sequences by Uniprot (yellow) and Phobius (orange) are depicted. ( B ) Table of experimental and calculated differences of two mGPDH forms. Experimental difference ( n = 5) was determined by the Molecular Weight Analysis tool (Image Lab software, Bio-Rad). ( C ) Cell lysates (50 μg protein) of HEK293 cells overexpressed with FLAG in different places of the GPD2 sequence were separated on SDS-PAGE (Hoefer System) and mGPDH forms were analyzed by Western blot using a specific antibody against FLAG ( n = 3). C-term—GPD2 with FLAG tag at the C-terminal, N-term—GPD2 with FLAG following initial start codon (N-term), 27AA—GPD2 with FLAG tag following 27th amino acid and 42AA—GPD2 with FLAG tag following 42nd amino acid. ( D ) Cell lysates (15 μg protein) of control PNT1A and cancer (LNCaP) cells were separated on SDS-PAGE and IMMP2L peptidase content was analyzed by Western blotting using a specific antibody ( n = 5). Actin was used as a loading control ( C–D ). Data represent the means ± S.D., ** p < 0.01.

Article Snippet: The normal human immortalized prostate epithelial cell line PNT1A (95012614, Merck/Sigma, Darmstadt, Germany) and androgen-sensitive human prostate cancer cell line LNCaP (kindly provided by Dr. Hodny, IMG CAS, Prague, Czech Republic) and human embryonic kidney cells (HEK293) were purchased from ATCC (CRL-1573, ATCC, Manassas, VA, USA).

Techniques: Molecular Weight, Software, Sequencing, SDS Page, Western Blot, FLAG-tag