Journal: EBioMedicine

Article Title: Severe metabolic alterations in liver cancer lead to ERK pathway activation and drug resistance

doi: 10.1016/j.ebiom.2020.102699

Figure Lengend Snippet: Severe metabolic alterations in HCC cells activate the ERK pathway. A. MTT proliferation assay of well-differentiated cells compared to poorly differentiated cell lines after 48 h of Gln deprivation. Box plot indicates mean ± S.E.M. of the proliferation data of 5 well-differentiated (HUH7, HEPG2, HEP3B, PLC/PRF/5, HUH1) and 5 poorly differentiated cells (SNU475, SNU398, SNU449, HLE, HLF) in glutamine (Gln)-free relative to complete media (CM). On the right, proliferation of patient-derived primary HCC cells, 72 h. B. Clonogenic assay with or without Gln. Fresh complete media (CM) or glutamine free media (- Gln), was introduced on Day 1 and 7 as indicated. The cells were stained with crystal violet after 14 days culture. C. Intracellular metabolite profile after 24 h culture with or without Gln. Error bars indicate mean ± S .E.M of 3 experiments. Cy. Cycle. Pyr – pyruvate, Lac – lactate, Cit – citrate, αKG – alpha ketoglutarate, Fum – fumarate, Mal – malate, Ser – serine, Met – methionine, Gly – glycine, Glu – glutamate, Asp – aspartate, Ala – alanine. D. Extracellular metabolite profile showing the proportion of metabolites consumption or secretion by HUH7 and HLE cells after Gln deprivation in serum-free media. The bars indicate mean ± S .E.M of the measured amount at 24, 28, 32 and 48 h after Gln deprivation relative to complete media, which is the baseline indicated with a broken line. Glc – glucose, E. Schematic of 13 C-glucose carbon labelling pattern in HUH7 and HLE cells deprived of extracellular Gln as deduced from isotope tracing. Broken line indicates the removal of glutamine from culture media. ↑ - increase, ↓ - decrease. Numbers in bracket indicate % of glucose-derived carbon that labelled the indicated metabolite in the respective cell line. m – the mass shift that contributed the most to the labelling. On the right, 13 C-glucose labelling of glutamine in HUH7 and HLE, respectively. F. Heatmap showing the expression pattern of 51 differentially regulated ERK pathway genes ( P <0.05) in Gln-deprived HLE cells. Underneath, MAPK pathway and other signalling-related pathway enrichment plots. G. Densitometric quantification of pERK level detected in western blots after glutamine deprivation at 24 h and 48 h (quantification from ≥4 experiments). H. Western blot showing pERK induction in HUH7 and HLE cells 7 days after culture in CM or Gln-free media. The western blot run included pAKT (shown). I. Western blot and densitometric quantification of pERK in two mouse hepatocyte isolates (48 h, 2 technical replicates). Error bars represent mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: SNU475 was a kind gift from Dr. Kathrin Woll (University of Heidelberg, Germany); SNU398 was from Dr. Francois Helle (University of Picardie Jules Verne, France), while SNU449 was from Dr. Cedric Coulouarn (INSERM, France).

Techniques: Proliferation Assay, Derivative Assay, Clonogenic Assay, Staining, Expressing, Western Blot