Supplementary Figure 2: AMPK is essential for optimal growth of primary GBM orthotopic xenografts in mice.

a, b, Luciferase imaging of mice to monitor tumour growth of GSC lines expressing AMPKβ1 or nontarget (NT) shRNA at indicated days. (n = 8-15 mice per line). Primary GSC lines AC17, 1123 were infected with NT or AMPKβ1 shRNA for two days and 10,000 Trypan blue negative live cells were transplanted into the cortex of NSG mice 48 hours after in vitro lentiviral transduction (when AMPK β1shRNA had negligible effect on viability). c, Quantification of tumour luminescence. Total range of tumour luminescence of mice was quantified three weeks after cell transplantation. (n = 20 mice / genotype). *p ≤ 0.0001. d, Luciferase imaging of mice to monitor tumour growth of a GSC line expressing AMPK α1α2 shRNA or nontarget (NT) shRNA at indicated days. (n = 6 mice per genotype). e, Kaplan-Meier survival data of mice in (d). f, Kaplan-Meier survival data of mice transplanted with U87EGFRvIII GBM serum line expressing NT or AMPK β1 shRNA. (n = 4 mice per genotype). g, Expression of cumate-inducible GFP in 293T cells. Scale bar 100μm. h, WB showing expression of cumate-inducible mouse AMPKβ1 (subcloned in the SparQ vector) in 293T cells. i, IHC pf pAMPK and pACC in NT and AMPKβ1 shRNA expressing tumours at indicated days. (T = tumour; N = normal brain). Scale bar 100 μm. j, Q-RTPCR of human AMPK β1 (using human-specific primers) in tumour cells isolated from NT shRNA or AMPKβ1 shRNA tumours. (n = 3). *p = 0.0007. k, WB of AMPKβ1/β2 and pAMPK in tumour-derived cells as in (j). Error bars; mean +/- S.D. Statistical significance in (c, j); two-tailed t-test. n values represent independent experiments. Source data are available in Supplementary Table 4. All western blots represent data from 2 (h) or 3 (k) independent repeats. Unprocessed blots are shown in Supplementary Fig. 9.