The Journal of Experimental Medicine

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1 [ID]FIGS1[/ID] [ID]FIGS2[/ID] [ID]FIGS3[/ID] [ID]FIGS4[/ID] [ID]FIGS5[/ID] [ID]FIGS6[/ID] [ID]TBLS1[/ID] [ID]TBLS2[/ID] SUPPLEMENTAL MATERIAL Wolf et al., The Journal of Experimental Medicine Figure S1. Expression of HK2, mutant IDH1 and apoptotic index in GBMs. (A) Immunohistochemistry for mutant IDH1R132H in the 12 GBM specimens from Fig. 1 A, as well as a low grade astrocytoma. Bars, 30 μm. (B) Apoptotic index in the center and periphery of 25 GBM samples measured by TUNEL immunohistochemistry. Graph depicts semiquantitative analysis of TUNEL-positive nuclei/total nuclei. Bar, 50 μm. (C) Hematoxylin and eosin and HK2 immunohistochemistry of representative perinecrotic center of GBM specimen, infiltrating periphery of GBM, LGA, and normal brain. Bar, 100 μm. (D) HK2 immunohistochemistry in LGAs and HGAs derived from GFAP: V12 Ha-Ras transgenic mice. S1

2 Figure S2. Inhibition of HK2 results in decreased cell viability. (A) Cell viability was in U87 and U373 cells transfected with scramble or indicated HK2 sirna (50 nm) measured by SRB assay over 5 d incubated under normoxic conditions or (B) 2% hypoxia. (C) Western blot of HK2 in U87 and U373 transfected 72 h prior with either HK2 sirna (50 nm) or scramble (scr) sirna (50 nm) and incubated under 2% hypoxia for 24 h. (D) Annexin V-PI flow cytometry in U87 and U373 cells transfected with HK2 sirna or scramble sirna 72 h prior, followed by 24 h of hypoxia. The percentages of cells in each quadrant are included. experiments represent mean ± SEM of three independent experiments. *, P < S2 Hexokinase 2 in glioblastoma multiforme Wolf et al.

3 Figure S3. Inhibition of HK2 in GBM explants reduces proliferation and sensitizes to radiation and TMZ. (A) Table depicting relevant genetic alterations in U87, U373, GBM6, and GBM8 cells. MGMT is a DNA damage repair enzyme important in the resistance to the chemotherapeutic TMZ. (B) Western blot of HK2 in GBM 6 and GBM 8 cells transfected 72 h prior with either HK2 sirna (25 nm) or scramble (scr) sirna (25 nm). (C) Fold change in BrdU incorporation in GBM6 and GBM8 cells transfected with scramble or HK2siRNA 7 d prior and treated with 5 Gy radiation or 24 h of TMZ. (D) Caspase 3 and 7 activity in GBM6 and GBM8 cells transfected with scramble or HK2siRNA 7 d prior and treated with 5 Gy radiation or 24 h of TMZ. Caspase 3 and 7 activity was measured using a fluorometric assay quantifying DEVD cleavage by caspase 3 and 7 quantified as fold difference in RFU (excitation at 498 nm, emission at 521 nm). (E) Fold difference in cell viability in U87 cells, U87 + PTEN cells, and U138 cells treated with 25 or 50 nm of HK2siRNA 72 h prior. BrdU, caspase, and cell viability assays were performed in triplicate and graphs show mean ± SEM of three independent experiments. *, P < 0.05; **, P < S3

4 Figure S4. Mitochondrial structure and count in U87 cells depleted of HK2, HK1, or PKM2. (A) Representative images of transmission electron microscopy of U87 cells transfected with scramble shrna, HK2shRNA1, or HK2shRNA2. Bar, 500 nm. (B) Representative images of live U87 cells from panel A, as well as HK1shRNA and PKM2shRNA stained with MitoTracker Deep Red. Bar, 16 μm. S4 Hexokinase 2 in glioblastoma multiforme Wolf et al.

5 Figure S5. Growth factor PI3K-AKT signaling is important for localization of HK2, but not HK1, to outer mitochondrial membrane and necessary for the growth promoting effects of HK2. (A) Immunofluorescence of U87 cells transfected with HK2-GFP or HK1-GFP under starved or EGF-stimulated conditions, or treated with EGFR tyrosine kinase inhibitor AG1478 or AKT inhibitor VIII. U87 cells with PTEN reintroduced were transfected with HK2-GFP, and colocalization with mitochondria was measured. U87 cells were also transfected truncated HK2-GFP that does not localize to the mitochondria (T2-GFP). Mitochondria are labeled with MitoTracker Deep Red (blue). Bar, 16 μm. Mitochondrial colocalization is seen in white in merged images. The percentage colocalization was measured across 5 10 cells in 2 independent experiments. (B) Western blot of HK2, pakt, and AKT in U87 and U343 cells. (C) Total HK activity in U343 cells transfected with HK2-GFP or HK1-GFP, normalized to control GFP transfected cells. HK assay was performed in triplicate and graph shows mean ± SEM of three independent experiments. (D) Western blot of HK2 in U87 cells treated with the protein inhibitor cycloheximide (CHX) in the presence or absence of EGF stimulation. S5

6 Figure S6. Stable loss of HK2 decreases in vivo GBM growth in a subcutaneous xenograft model and decreases HIF1 stability and VEGF expression. (A) Mean tumor volume of NOD-SCID mice injected subcutaneously with U87 cells expressing scramble shrna ( n = 7) 4 wk prior or HK- 2shRNA (1 and 2, n = 8) 8 wk prior (P < 0.001). (B) Histopathology of U87 scramble shrna and U87HK2shRNA subcutaneous tumors including hematoxylin and eosin staining (bar, 200 μm) and antibodies specific to HK2 (bar, 100 μm), HIF1 (bar, 100 μm), cleaved caspase 3 (bar, 100 μm). Necrosis (N) is labeled on U87 scramble shrna tumors. Arrows show positive nuclei for HIF1 or cleaved caspase 3. (C) HIF1 mrna was measured by qrt-pcr in U87 cells transfected with scramble shrna, HK2shRNA1, or HK2shRNA. (D) Western blot of HK2, HIF1, PHD2, and VEGF in U87 cells transfected with scramble shrna, U87HK2shRNA1, and U87HK2shRNA2. Table S1. Pairwise comparison in expression of HK2 transcript across GBM subtypes using significance analysis of microarrays NL CL Mes PN CL Mes PN NL Mes Mesvs PN Mes vs. NL CL, classical; Mes, mesenchymal; NL, neural; PN, proneural. Verhaak et al., Mesvs CL Nesvs S6 Hexokinase 2 in glioblastoma multiforme Wolf et al.

7 Table S2. Primary antibodies used for immunohistochemical staining with dilutions Primary antibody Dilution 1 h room temperature or 4 C o/n Manufacturer vwf 1:400 1 h RT DAKO MIB1 1:100 1 h RT DAKO HK2 1:100 4 o/n Cell Signaling Technology HK1 1:500 4 o/n Millipore HIF1 1:50 4 o/n Laboratory Vision VEGF 1:50 1 h RT Santa Cruz Biotechnology, Inc. Cleaved caspase 3 1:50 4 o/n Cell Signaling Technology REFERENCE Verhaak, R.G., K.A. Hoadley, E. Purdom, V. Wang, Y. Qi, M.D. Wilkerson, C.R. Miller, L. Ding, T. Golub, J.P. Mesirov, et al Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 17 : doi: /j.ccr S7

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