Figure 8.

Astrocytes feed with Gln GBM cells. (a, b) Astrocytes were incubated in SMEM for 6 days with 0, 0.1, 0.3, 0.65, 1, 2, 4mM Gln (a) or for the indicated times with 0 and 0.65mM Gln (b). (c) Astrocytes derived from two independent extractions, and cell lines, were incubated for 3 days +/− Gln and protein expression assessed. Unprocessed scans of western blots are shown in Supplementary Figure 8. (d-j) Astrocytes were incubated in SMEM for 24h in the presence of 5.56mM Glucose (¹³C₆ or ¹³C₀), 0.65mM Gln (¹³C₅ or ¹³C₀), 0.8mM ¹⁵NH₄⁺, and 1mM MSO as indicated. Secretion/consumption rates (positive/negative bars respectively) are shown for Gln (d) and Glu (e). Intracellular levels of Gln (f) and Glu (g) isotopologues are reported as % of control (total of isotopologues in Gln fed conditions). The intracellular isotopologues of Gln (h), Glu (i), AMP (j) are shown as % of control (total isotopologues in the presence of Gln and ¹⁵NH₄. (k) Astrocytes were incubated for 6 days +/− 0.65mM Gln, and 1mM MSO and counted. (l) Astrocytes were grown to confluence in multi-well plates. iRFP₄ cells were seeded in wells +/− astrocytes, and +/− Gln. The fluorescence of iRFP4 cells in representative wells is shown. The experiment was performed twice with comparable results. (m) Astrocytes were grown to confluence in multi-well plates. iRFP₄ cells seeded in transwell inserts were co-cultured +/− astrocytes, +/−Gln, and +/− Erwinase (Erw) as indicated. Fluorescence of iRFP4 cells in representative inserts is shown. At day 5 astrocytes were stained with sulphorodamine-B and the fluorescence of representative wells is shown. (n) Quantification of the iRFP₄ fluorescence as described for (m). (a, b, d, e, f, g, h, i, j, k, n) Data derive from one experiment performed twice. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.