Fig. 2.

V-ATPase levels determine glioma growth in vivo. a–d) Down-regulation of VhaPPA1–1 prevents tumor growth in brains of Drosophila larvae. a) Single confocal medial sections of whole brains from 3rd instar larvae. Dorsal view, anterior up. Glial tissue is labeled by a membrane-targeted form of GFP (CD8GFP; green) expressed under the glial specific promoter of reverse polarity (repo) gene. Note the increase in CD8GFP expression and brain size in larvae expressing ΔEGFR and Pi3K92E-CAAX under repo (repo> CD8GFP ΔEGFR Pi3K92E-CAAX), which is prevented by downregulating VhaPPA1–1 (repo> CD8GFP ΔEGFR Pi3K92E-CAAX VhaPPA1–1). Scale bar, 100 μm. b) VhaPPA1–1 mRNA was measured in brains of 3rd instar larvae by qPCR. Bars, means ± s.d. of three independent experiments. c,d) Surface area (c) or GFP pixel intensity (d) quantification of brain sections (>6 brains per genotype). Bars, mean ± SEM; **, p < 0·01; ***, p < 0·001 (Kruskal-Wallis test with Dunn's post-test). e, f) GBM-derived neurospheres (NS) with low or high levels of V-ATPase V1G1 were stably transfected with a luciferase construct and injected intracranially into nude mice (n = 8 per group). Tumor growth was monitored over time (up to Day 109) by measuring luciferase emission (BL counts). **, p < 0·01; ***, p < 0·001 (Kruskal-Wallis test with Dunn's post-test). Dots, mean ± SEM (n = 13 per group). g) Representative hematoxylin and eosin (H&E) and STEM121 stains of brains from mice injected with V-ATPase V1G1 low- (left panels) or high- (right panels) NS at the time of harvesting (Day 109). h) Glioma cell proliferation (Ki67 staining) in the brains of indicated mice. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)