B. C. Staurosporine BEZ235 DMSO. -actin. Suppl. Fig. 1 BEZ235 and BGT226 inhibit phosphorylation of Akt and downstream targets of PI3K and mtor.

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1 DMSO Staurosporine BGT226 A. B. C. BGT226 P-Akt hrs. P-Akt nm PS6 P-4E-BP1 -actin C-Caspase3 -actin PS6 P-4E-BP1 -actin Suppl. Fig. 1 and BGT226 inhibit phosphorylation of Akt and downstream targets of PI3K and mtor. (A) SQ20B cells were seeded and allowed to attach prior to treatment with (50 nm) for the times indicated prior to harvesting. (B) SQ20B cells were treated with either (50 nm) or BGT226 (50 nm) for 16 hours prior to harvesting. Staurosporine (1uM for 16hr) was used as a positive control for apoptosis. Arrows denote cleaved form of caspase3. (C) SQ20B cells were treated with increasing concentrations of BGT226 for 16 hours prior to harvesting. Western blotting was performed using antibodies as indicated.

2 DMSO 1hr 2hr 4hr 8hr 16hr DMSO 1hr 2hr 4hr 8hr 16hr OCR(pMoles/min)/mg protein OCR(pMoles/min)/mg protein BGT p = ns * DMSO N 1H O 2 HR Q 4HR S 8 HR U 16 HR W p = ns ** DMSO O 1HR T 2HR W 4HR Y 8HR X 16HR P Suppl. Fig. 2 Reduction of O2 consumption in vitro as a function of duration of drug incubation. SQ20B cell were seeded into flux analyzer plates and allowed to attach. Either or BGT226 was added for varying amounts of time, as indicated, prior to measurement of OCR. Comparison of OCR at each time point (T=1, 2, 4, 8, 16 hrs.) was made to DMSO control (ns: p is non-significant, *: p < 0.04; ** p < 0.028)

3 Percent of max Percent of max A. B Control 91.7 ± ± Control Mean fluorescent Intensity Mean fluorescent Intensity Suppl. Fig. 3 Effect of mitochondrial mass and membrane potential. SQ20B cells (1X10 6 ) were seeded into dishes and treated for 16 hrs with (50 nm). Following drug treatment, cells and controls were stained with either (A) MitoTracker Green (50nM) or (B) Mito-Tracker Red (50nM) for 30 min. After MitoTracker staining cells were trypsinized and stained with Annexin V for viability and DAPI for analysis on the LSRII. Flow analysis showed no difference between controls or drug-treated cells. Statistical analysis was conducted using student-t test using 3 independent experiments (p > 0.1 for both).

4 Fold change (COX1/PNC1) 2 n.s. control NVP control Suppl. Fig. 4 had no effect on mitochondrial mass measured by RT-qPCR. Total DNA was isolated as described before (Miller, S.A, Dykes, D.D. and Polesky, H.F. (1988) Nucleic Acids Res. 16, 1215). Briefly, cells were lysed in lysis buffer (100mM Tris-HCL (ph 8.5), 5mM EDTA, 0.2% SDS and 10 mg/ml proteinase K) at 55 1C for 2 h. Total DNA was extracted using ethanol precipitation. Owing to the absence of introns in the mitochondrial genome, mtdna was assessed using primer pairs that are complementary to sequences spanning two genes: Cox1 for mtdna F and Cox2 for mtdna R (see Table S1 in Supplementary Figures). The levels of nuclear DNA were measured using two pairs of primers located on either one exon and one intron of pnc1 gene or two introns of the actin gene (pnc1 exon4 and intron4, and a-actin intron3 and intron4 for DNA F, DNA R, actdna F and actdna R, respectively). The data are presented as ratio of COX1 to PNC1 with control normalized to 1.0. Data are presented as mean of 3 independent experiments sd. Statistical analysis was conducted by student-t test (p > 0.1). Sequence of primers used are shown in table above.

5 control BGT226 P-Akt Tot-Akt P-S6 Tot-S6 P-4E-BP1 Tot-4E-BP1 E1 E2 E2/E3bp Tot PDH -Actin Suppl. Fig. 5 and BGT226 do not alter expression of E, E2 or E2/E3bp. SQ20B cells were treated with either or BGT226 for 16hrs, then samples were harvested and immunoblotting performed.

6 OCR (pmoles/min)/mg protein A. B. C. SQ20B SQ20B FaDu OCR(pMoles/min)/mg protein G2- DMSO G4 - GDC-0068 OCR(pMoles/min)/mg protein G1- DMSO G5 - GDC-0980 DMSO BGT226 Suppl. Fig. 6 GDC-0068 and GDC-0980 decrease O 2 consumption in response to inhibitors. SQ20B cells were seeded into Seahorse Analyzer plates and allowed to attach before either (A) 5 µm GDC-0068 or (B) 1 µm GDC-0980 was added for 16 hours prior to measurement of OCR. Vertical lines labeled A, B, and C indicate times respectively when oligomycin, FCCP, or rotenone was added. (C) FADU cells were seeded into Seahorse Analyzer plates and allowed to attach before or BGT226 was added for 16 hours prior to measurement of OCR. Vertical lines labeled A, B, and C indicate times respectively when oligomycin, FCCP, or rotenone was added.

7 OCR(pMoles/min)/mg protein OCR (pmoles/min)/mg protein A. U251-PTEN B. U251-C124S U251-mutPTEN U251-mutPTEN + DOX B. Suppl. Fig. 7 Addition of doxycycline to u251-pten but not U251-C124S reduces O 2 consumption. U251MG cells engineered to be inducible for either wild-type PTEN or phosphatase-dead PTEN (C124S) were exposed to doxycycline (1µg/ml). (A) U251-PTEN cells were seeded into Seahorse plate and allowed to attach prior to treatment with doxycycline for 16h. Seahorse plates were then monitored for their O 2 consumption. Lines A, B, C represent times when oligomycin, FCCP, and rotenone were added respectively. (B) same as (A) except U251-C124S cells were used.

8 OCR(pMoles/min)/mg protein OCR(pMoles/min)/mg protein A. B. A B C G2-Scramble G4-PDH sirna G5- G6-BGT226 Suppl. Fig. 8 Effect of dicholoroacetate (DCA) treatment on OCR. Cells were seeded into Seahorse analyzer plates and allowed to attach. Cells were treated with 20 mm dichloroacetate (DCA) for one hour prior to being treated for 16 hours with either or BGT226. OCR was then determined using Seahorse Analyzer. Vertical lines labeled A, B, and C indicate times respectively when Oligomycin, FCCP, or rotenone was added.

9 DMSO DMSO + pbabe- neo pbabe- neo+ WtE1 WtE1 +BEZ25 1S 1S + 3S 3S + PDH (S232) PDH (S293) ** * PDH (S300) Tot-PDH -actin lane: Suppl. Fig. 9. Ectopic expression of PDH-E1a mutants that cannot be phosphorylated blunts -mediated increase in endogenous PDH phosphorylation. SQ20B cells were infected with a retrovirus expressing either wild type PDH-E1 or PDH-E1 expressing a single 1S (S232A) OR triple 3S (S232A, S293A,S300A) mutation. 72 hours later cells were treated with 50 nm. 16hrs later cells were harvested for Western blot analysis. (*) denotes endogenous PDH (S232) and (**) denotes expressed form of PDH (S232).

10 Name Sequence mtdna F 5'-CCACCCTACCACACATTCG-3' mtdna R 5'-GGATCGTTGACCTCGTCTGT-3 PNC1 DNA F 5'-TGTTACTCCAAAGCCAAAGAGC-3' PNC1 DNA R 5'-TCTATCTATATCATTTGATGGGCAAAC-3 Table S1: Sequence of primers used to measure the levels of the mitochondrial gene COX1.