Journal: Scientific Reports

Article Title: Role of the Transforming Growth Factor-β in regulating hepatocellular carcinoma oxidative metabolism

doi: 10.1038/s41598-017-12837-y

Figure Lengend Snippet: Glycolytic and oxidative metabolism in different HCC cell lines. (a) Immunofluorescence analysis of E-cadherin (green) and Vimentin (green). DAPI (blue). Scale bar represents 50 μM. (b) mRNA expression levels of TGF-β detected by qRT-PCR and normalized to PLC/PRF/5. Mean ± SD (n = 3). (c) OCR was analyzed at basal condition by high resolution respirometry (Oxygraph 2k) in DMEM supplemented with 10% FBS in 5 × 10 5 cells in suspension. Results are represented as OCR pmol/seg/ml of the first 10 min of respiration after stabilization. (d,e) Lactate production and glucose consumption was analyzed after 48 hours of cell culture in DMEM supplemented with 10% FBS and normalized to cell count. (f) Cells were treated with 2-DG in a range of concentration (0–10 mM). Cell viability was analyzed after 72 hours by crystal violet staining and normalized to un-treated control. (g) Cells were treated with metformin in a range of concentration (0–10 mM). Cell viability was analyzed after 72 hours by crystal violet staining and normalized to un-treated control. Mean ± SD (n = 3, p values are explained in the table below figure).

Article Snippet: For glutamine labeling DMEM medium with 25 mM glucose containing 10% dialyzed FBS and 4 mM fully labeled glutamine (Cambridge Isotope Laboratories Inc., product reference CLM-1822-H-PK) was used.

Techniques: Immunofluorescence, Expressing, Quantitative RT-PCR, Suspension, Cell Culture, Cell Counting, Concentration Assay, Staining, Control

Journal: Scientific Reports

Article Title: Role of the Transforming Growth Factor-β in regulating hepatocellular carcinoma oxidative metabolism

doi: 10.1038/s41598-017-12837-y

Figure Lengend Snippet: Metabolomic and transcriptomic analysis of PLC/PRF/5 and SNU449 cells: differences in the TCA cycle and glutamine metabolism. (a ) Left: Schematic diagram of the TCA cycle as presented in the KEGG database ( http://www.genome.jp/kegg/ ). The level of metabolites is depicted by a box plot with whiskers (min to max). Welch’s two-sample t -test was used to identify metabolites that differed significantly between experimental groups (n = 5 for each group). *p < 0.05, **p < 0.01, ***p < 0.001. Right: Expression of FH and selected genes related to the glutamine metabolism pathway. Values < 1 indicate lower expression and values > 1 indicate higher expression, SNU449 as compared to PLC/PRF/5. (n = 3, p value indicated in the right column). (b) Metabolites from the glutamine/glutamate pathway presented in fold comparing SNU449 to PLC/PRF/5. Welch’s two-sample t -test was used to identify metabolites that differed significantly between experimental groups (n = 5 for each group, p value indicated in the right column). (c) PLC/PRF/5 cells and SNU449 were cultivated in DMEM medium (25 mM glucose) without glutamine supplemented with 10% FBS. Cell viability was analyzed after 24, 48 and 72 hours by crystal violet staining and normalized to control (2 mM glutamine). Mean ± SD (n = 3), **p < 0.01, ***p < 0.001.

Article Snippet: For glutamine labeling DMEM medium with 25 mM glucose containing 10% dialyzed FBS and 4 mM fully labeled glutamine (Cambridge Isotope Laboratories Inc., product reference CLM-1822-H-PK) was used.

Techniques: Expressing, Staining, Control

Journal: Scientific Reports

Article Title: Role of the Transforming Growth Factor-β in regulating hepatocellular carcinoma oxidative metabolism

doi: 10.1038/s41598-017-12837-y

Figure Lengend Snippet: 13C isotopomer distribution from fully labeled glutamine/glucose. (a) PLC/PRF/5 and SNU449 cells were exposed to 4 mM fully labeled glutamine (U-13C5-Glutamine) in a medium containing 25 mM glucose and 10% dialyzed FBS. (b – d ) PLC/PRF/5 and SNU449 cells were exposed to 25 mM fully labeled glucose (U-13C6-Glucose) in a medium containing 4 mM glutamine and 10% dialyzed FBS. As 13 C from glutamine/glucose is distributed among various metabolites, their mass increases proportionally to the number of incorporated carbons. This increase in mass was detected by GC-MS. Isotope distribution in metabolites is marked as m + x, where the m stands for natural mass of the metabolite and the x indicates number of incorporated 13 C carbons. Mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For glutamine labeling DMEM medium with 25 mM glucose containing 10% dialyzed FBS and 4 mM fully labeled glutamine (Cambridge Isotope Laboratories Inc., product reference CLM-1822-H-PK) was used.

Techniques: Labeling, Gas Chromatography-Mass Spectrometry

Journal: Scientific Reports

Article Title: Role of the Transforming Growth Factor-β in regulating hepatocellular carcinoma oxidative metabolism

doi: 10.1038/s41598-017-12837-y

Figure Lengend Snippet: Seahorse analysis of OXPHOS and glycolysis in HCC cells. Role of the TGF-β pathway. (a,b) OCR normalized to protein content in PLC, TβT-PLC (a) and SNU449sh-, SNU449shTβRI cells (b) incubated 30 minutes prior experiment in XF assay medium supplemented with 5 mM glucose and 2 mM glutamine and consecutively injected with oligomycin (1 μM), FCCP (1.5 μM), antimycin (1 μM) and rotenone (1 μM). Continuous OCR values (pmoles/min/µg protein) are shown. Mitochondrial functions were analysed as explained in Supplementary materials and methods. The % of ATP-linked OCR was calculated as ATP-linked OCR/basal OCR. Mean ± SEM (n at least 6 from three independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001. (c,d) ECAR in PLC, TβT-PLC (c) and SNU449sh-, SNU449shTβRI cells (d) incubated 30 minutes prior experiment in XF assay medium supplemented with 2 mM glutamine and consecutively injected with glucose (10 mM), oligomycin (1 μM) and deoxyglucose (50 mM). Mean ± SEM (n at least 9 from three independent experiments). Continuous ECAR values (mpH/min/µg protein) are shown. Glycolytic functions were analysed as explained in Supplementary materials and methods. Mean ± SEM (n = 9 from three independent experiments). Glucose consumption (mg/10 5 cells) was measured after 48 hours of culture in DMEM supplemented with 10% FBS and normalized to cell number. Mean ± SD (n = 3).

Article Snippet: For glutamine labeling DMEM medium with 25 mM glucose containing 10% dialyzed FBS and 4 mM fully labeled glutamine (Cambridge Isotope Laboratories Inc., product reference CLM-1822-H-PK) was used.

Techniques: Incubation, XF Assay, Injection