Fig 1: A metabolic shift from aerobic glycolysis towards amino acid metabolism inhibits proliferation but enhances goblet cell differentiation of CRC cell lines in vitro. (a,b) MC-38 cells were incubated in culture media containing increasing concentrations of D-glucose (0–25 mM) in the presence of 1%, 10%, or 20% NEAA. After 72 h, cell viability was determined by neutral red cytotoxicity assay. (c) Representative images of immunohistochemistry staining of paraffin-embedded MC-38 cells after 48 h cultivation under standard (25 mM glucose + 1% NEAA) or glucose-free (0 mM glucose + 20% NEAA) conditions, utilizing an antibody cocktail against OXPHOS complexes I–V. (d) MC-38 cells were incubated in standard culture medium (25 mM glucose, 1% NEAA) or in glucose-free plus 20% NEAA medium for 48 h. Expression of OXPHOS complexes II, III, and V or LC3B were analyzed by Western blot experiments. Alpha-Tubulin expression served as a loading control (representative images of n = 3 independent experiments). (e) MC-38 cells were incubated in a standard culture medium (25 mM glucose, 1% NEAA) or glucose-free plus 20% NEAA medium for 24 or 48 h. Caspase-1 mRNA expression was quantified by qPCR experiments and related to Ubc mRNA expression (left panel; n = 3 independent experiments). Caspase-1 protein expression after 48 h of incubation was analyzed by Western blot experiments. Alpha-Tubulin expression served as a loading control (right panel; representative images of n = 3 independent experiments). (f) Protein levels of phosphorylated AKT and AKT were determined after 48 h of incubation of MC-38 cells under indicated culture conditions by Western blot experiments. ß-actin expression served as a loading control. Representative images of n = 3 independent experiments. (g) HT29-MTX cells were incubated for 72 h under indicated culture conditions and cell count was determined. Mean ± SEM of four independent experiments is presented. (h,i) MC-38 cells were incubated under indicated culture conditions. After 72 h, L-lactate levels were quantified in cell culture supernatants. (j) L-lactate release by HT29-MTX cells after 72 h of incubation under indicated culture conditions. L-lactate concentrations were related to the respective cell count presented in (g). Mean ± SEM of three independent experiments is presented. (k) MC-38 cells were incubated in standard culture medium (25 mM glucose, 1% NEAA) or in glucose-free plus 20% NEAA medium for 24 or 48 h. Klf4, Muc2, or Ubc mRNA expression was quantified by qPCR experiments (n = 3 independent experiments). ?CT values were calculated by normalization of Klf4 or Muc2 to Ubc and related to values received from standard culture conditions at 24 h. Muc2 secretion was analyzed after 72 h of incubation in cell culture supernatants from respective cells by ELISA experiments. Data are related to cell viability determined by neutral-red cytotoxicity assay and presented as mean ± SEM. (l) Muc5AC release by HT29-MTX cells was measured after 72 h of incubation and related to respective cell counts. Values were normalized to Muc5AC secretion observed for standard culture conditions. * p = 0.05, ** p = 0.01. *** p = 0.001. AU = arbitrary units.