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The BRAF inhibitor vemurafenib activates mitochondrial
metabolism and inhibits hyperpolarized pyruvate-lactate
exchange in BRAF mutant human melanoma cells
SUPPLEMENTARY MATERIAL AND METHODS
Western blotting
The following primary antibodies were used: anti-MEK1/2, anti-ERK1/2 (total and
phosphorylated), anti-hexokinase II (HKII), anti-acyl-CoA dehydrogenase family, member 9
(ACAD9) (Cell Signaling Technology; Danvers, MA, USA), anti-lactate dehydrogenase-A
(LDH-A),
anti-3
phosphoglycerate
dehydrogenase
(3PGDH),
anti-monocarboxylate
transporter 4 (MCT4) (Santa Cruz Biotechnology; Santa Cruz, CA, USA), antimonocarboxylate
transporter
1
(MCT1)
(Sigma-Aldrich;
Gillingham,
UK),
anti-
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore, Nottingham, UK), antiacetyl-CoA carboxylse (Cell Signaling Technology; Danvers, MA, USA) and anti-phosphoATP-citrate Lyase (P-ACL) (Cell Signaling Technology; Danvers, MA, USA). The
secondary antibodies used were anti-rabbit (GE Healthcare Life Sciences; Buckinghamshire,
UK), anti-goat (Santa Cruz Biotechnology; Santa Cruz, CA, USA) and anti-mouse
Immunoglobulins/HRP (Dako A/S, Glostrup, Denmark).
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Quantitative real-time PCR (qRT-PCR)
The
Hs00606086_m1,
Hs00855332_g1,
Hs01561850_m1,
Hs00198333_m1,
Hs00606568_gH, Hs00795278_mH, Hs01580591_m1, Hs005599398_m1, Hs01855675_s1,
Hs00998733_m1 and Hs01126940_gH assays were used for the HKII, LHDH-A, PDK-1,
PHGDH, GCAT, PSAT-1, GLDC, PC, IDH-1, GLS and ISYNA-1 genes respectively (gene
specific primers and FAM labeled probe) multiplexed with the endogenous control assay for
β-ACTIN gene (β-ACTIN-specific primers and VIC/TAMARA-labeled probe; Applied
Biosystems). mRNA levels of each gene were determined for each sample in the same well
on the ABI 7900HT relative to those of β-ACTIN and data processed by expressing changes
as the differences in the threshold cycle (2-∆∆Ct), inversely proportional to the amount of
target mRNA per sample.
Reactive oxygen species (ROS) production assay
Detection of ROS was assessed using a DCFDA-based cellular reactive oxygen species
detection assay kit (Abcam, Cambridge, UK) using manufacturer instructions. 50µM TertButyl Hydrogen Peroxide (TBHP) was used as a positive control following the kit
recommendations. For the negative control, N-acetyl cysteine (NAC) was applied 24h before
acquiring the data, as it has been reported to be a ROS inhibitor (1). Two hours before
reading, TBHP was added to the negative control in order to test the efficacy of ROS
inhibition.
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NMR metabolic analyses of cells
Cells were extracted with a methanol-chloroform-water method and lyophilized samples
of the aqueous fraction reconstituted in 540 µL of D2O containing 5% of 3-(trimethylsilyl)
propionic-2,2,3,3-d4 acid (TSP, internal reference and quantification standard for 1H NMR).
Following 1H NMR acquisition, EDTA and methylenediphosphonic acid (MDPA, internal
standard) were added to a final concentration of 10 and 0.86 mM, respectively, at pH 8.2 for
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P NMR analysis. Lipid fractions were re-suspended after chloroform evaporation in a 0.03
% (v/v) tetramethylsilane (TMS, internal reference) d-chloroform solution. Media fractions
were collected from control and treated cells, centrifuged to discard debris and 1ml of each
freeze-dried and re-suspended in D2O-TSP.
NMR data acquisition and processing
For 1H NMR, a 30° flip angle, a 2s relaxation delay (RD), a spectral width of 13 ppm and
64 k data points were used under conditions of water signal suppression for aqueous samples.
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P NMR spectra were acquired using power gated composite pulse 1H decoupling, a 30° flip
angle, a 1s RD, a spectral width of 100 ppm and 32 k data points.
13
C NMR data were
acquired using power gated composite pulse 1H decoupling, 33 k data points, a 250 ppm
spectral width, a repetition time of 6 s and a 30° flip angle. A line broadening of 0.2 Hz was
applied for 1H, 2Hz for 31P spectra and 5 Hz for 13C spectra.
For hyperpolarized NMR experiments, data were acquired at 37oC using an inverse gated
pulse sequence, a sweep width of 150 ppm, covering the carbonyl region of the 13C spectrum,
8 k data points and a 5o flip angle, and spectra processed using Bruker Topspin v3.2.
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Measurements of ADP/ATP ratios
To determine ADP/ATP ratios, control and treated cells (2 µm vemurafenib, 24h) were
harvested and processed according to manufacturer’s instructions using a bioluminescent
ADP/ATP Ratio Assay kit (Abcam, UK).
SUPPLEMENTARY FIGURES
Suppl. Fig. S1: Cell proliferation is not significantly different using 25 mM or 5 mM glucose
medium after 24h or 48h of treatment. Cell numbers calculated in both DMSO and vemurafenib
treated samples growing for 24 and 48h under vemurafenib treatment in media containing 25 mM or 5
mM glucose. The difference in cell number between DMSO and vemurafenib samples is significant
after 48h of treatment, with 5 mM glucose producing similar effects to 25 mM glucose. *P < 0.05.
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Suppl. Fig. S2: BRAF mutant cells maintain their bioenergetics level under BRAF inhibition. A)
A 31P NMR spectrum obtained from the aqueous fraction of control WM266.4 BRAF mutant
melanoma cancer cell extracts. Metabolites detectable in the spectrum: PC, phosphocholine; Pi,
inorganic phosphate; GPE, glycerophosphoethanolamine; GPC, glycerophosphocholine; PCre,
phosphocreatine; α-NTP, β-NTP, and γ-NTP, nucleotide triphosphates. B) % change in the metabolic
analysis of treated cells with respect to the controls. C) ADP/ATP ratio calculated in control and
treated cells using a bioluminescence assay (p = 0.53).
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Suppl. Fig. S3: BRAF mutant WM266.4 cells show increased ROS levels under BRAF
inhibition. TBHP (50 µM) and NAC (3 mM) + TBHP (50 µM) were used as positive and negative
controls, respectively. Vemurafenib concentrations were 0.25, 0.5, 1 and 2 µM. *P < 0.05; ** P ≤
0.01.
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Suppl. Fig. S4: Changes in cell counts in WM266.4 and SKMEL28 control and treated samples
in the 4 nutrient-restricted conditions. Cell numbers were calculated in both DMSO and
vemurafenib treated samples of WM2664 (A) and SKMEL28 (B) cell lines after 24 and 48h of
treatment. Dotted line indicates 100% (cell seeding density). C) A representative example of total
PARP expression analysed in WM266.4 cells after 48h of treatment in the 4 media conditions shown
in A). No signs of apoptosis were detected in any case. Legend: 1) 1 mM glucose; 2) 1 mM glucose
without glutamine; 3) 1 mM glucose without glutamine and pyruvate; 4) 5 mM glucose; C) control;
T) vemurafenib.
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Suppl. Fig. S5: Changes in cell cycle distribution in control and vemurafenib-treated WM266.4
cell samples in the 4 different nutrient conditions. The percentage of cells in each phase of the cell
cycle (G1, S or G2) was calculated for control and treated samples in 4 different media conditions: A)
5 mM glucose, B) 1 mM glucose, C) 1 mM glucose without glutamine and D) 1mM glucose without
glutamine and pyruvate. The characteristic G1/S cell cycle arrest observed with vemurafenib exposure
in BRAF mutant WM266.4 cells in replete media (5mM glucose) is gradually lost with sequential
removal of nutrients.
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Suppl. Fig. S6: Cell lipid content detection by 1H NMR. Schematic representation of a
phospholipid molecule showing the main protons detected in the 1H NMR spectra (marked in bold).
These chemical components correlate with the resonances analysed in Figure 1D (x axis in the graph).
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