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. 2016 Sep 28;7(45):73292–73308. doi: 10.18632/oncotarget.12320

Figure 2. Physiological citrulline concentration supports CRC cell growth neither in monolayer nor in spheroid culture despite upregulation of ASS1 protein expression.

Figure 2

(A) Representative Western blots of protein extracts from monolayer and spheroid cultures of HCT-116 cells harvested before (day 0) or after a 3-day exposure to arginine-free (−Arg) medium alone or supplemented with the indicated concentrations of citrulline (Cit) are shown. Total protein extracts were probed with an anti-ASS1 antibody. α-TUBULIN was used as loading control. (B) HCT-116 or HT29 cells in monolayer or spheroid culture were incubated for the indicated periods of time in –Arg medium supplemented with Arg or Cit to final concentrations of 0.05 or 0.4 mM. Monolayer cell growth was assessed by the quantification of viable cells via an MTT assay in three independent experiments. Spheroid growth was determined by calculation of spheroid volume from the measured diameters of spheroids. Each data point presents the average volume (± SE) of at least 30 spheroids obtained in one experiment. This result was verified by an independent experimental series.