Fig. 1. PFKFB4 silencing reduces tumor size in vivo.

A PFKFB4 protein levels in luciferase expressing NCH421k GSCs stably transduced with doxycycline inducible shNT or shPFKFB4 on day 4 with or without doxycycline. α-tubulin was used as a protein loading control. B (Left) Representative images of NCH421k spheres 6 days after induction of shNT or shPFKFB4 with doxycycline. (Right) Percentage of living cells as determined by propidium iodide (PI) staining and FACS analysis 2 and 4 days after induction of shNT or shPFKFB4 with doxycycline (n = 3, mean ± SD, two sided t-test, *p value < 0.05, **p value< 0.01). C Bioluminescence signal of representative mice orthotopically transplanted with shNT and shPFKFB4 transduced cells at observation time-points 0, 1, 2 and 10, corresponding to 0, 4, 14 and 42 days, after starting administration of doxycycline. D Mean bioluminescence signal of mice transplanted with shNT transduced cells (black) and shPFKFB4 transduced cells (black dotted) after start of administration of doxycycline and mice transplanted with shPFKFB4 transduced cells which did not receive doxycycline (gray) at 10 different time points over a period of 35 days. Data are represented as mean ± standard error of the mean (SEM), two-sided t-test, *p value < 0.05, **p value < 0.01, ***p value < 0.001. E Survival of mice transplanted with shNT (black, n = 8) and shPFKFB4 transduced cells (black dotted, n = 7) after start of administration of doxycycline, and mice transplanted with shPFKFB4 transduced cells which did not receive doxycycline (gray, n = 7). Censored observations are represented by a vertical dash and represent mice which were sacrificed before they displayed any neuropathological symptoms for monitoring tumor growth by immunohistochemistry. Log rank test, ***p value < 0.001. F Representative H&E stained tissue sections of brains of a mouse from each group and PFKFB4 protein expression (below). Scale bar = 20 µm.