Fig. 6

TGF-β1 induced CAFs and glutamine metabolism between CAFs and B16F10 cells. (A) Optimal time for induction of CAFs formation. (B) Optimal concentration of TGF-β for induction of CAFs formation. (C) Quantification of different treatment times of α-SMA/GAPDH. (D) Quantification of different treatment concentration of TGF-β of α-SMA/GAPDH. (E) Representative images and (F)histograms of the effect of normal medium, NIH/3T3 CM and CAFs CM on the colony forming ability of B16F10 after 10 days of incubation. (G) Cell viability of different CM on B16F10 cell growth after 24 h of incubation. (H&I) B16F10, NIH/3T3 and CAFs were cultured in Gln-deficient medium for 6 h. CAFs CM and NIH/3T3 CM were taken to replace the glutamate-free medium of B16F10 and the colony-forming ability was verified after 10 days. B16F10 cultured in glutamate-free medium was used as a control experiment. Representative images(H) and histograms(I) of the effect of different CM on colony-forming capacity of B16F10. (J)Cell viability of B16F10 cells with different condition medium. (K) IHC stained images comparing GLUL protein expression between stromal and tumor compartments. Scale bars: 100 μm. CAFs CM is conditioned medium derived from CAFs, NIH/3T3 CM is conditioned medium derived from NIH/3T3. -Gln is the glutamine deprivation condition, -Glc is the glucose deprivation condition, and -Gln/Glc is the simultaneous glutamine and glucose deprivation condition. **p < 0.01, ***p < 0.001, ****p < 0.0001 (ANOVA test). All statistical data are expressed as means ± SD (n = 3)