Extended Data Figure 4. Effect of oxygen availability, nutrient availability and anchorage on reductive carboxylation.

a, Mass isotopologues of citrate in spheroids cultured with [U-¹³C]glutamine under 20% and 60% oxygen (n=4 cultures from two experiments). b, Effects of reducing extracellular glucose and glutamine concentrations on citrate mass isotopologues in monolayer cells cultured with [U-¹³C]glutamine (n=2 cultures from a representative experiment). c, Day 7 spheroids were allowed to attach to a conventional tissue culture dish for 24h, and mass isotopologues of citrate were analyzed with [U-¹³C]glutamine tracing (n=4 cultures from two experiments). Insets are photomicrographs of cells in each of the culture conditions. Scale bars, 200 µm. *p<0.05 comparing to monolayer, #p<0.05 comparing to spheroid, Welch's unequal variances t-test followed by multiple-comparison correction. All experiments were repeated 3 times or more.