Download The effects of severe hypoxia on glycolytic flux and enzyme activity

The effects of severe hypoxia on glycolytic flux and enzyme activity in a solid tumour model
Hannah Smith1, Mary Board1, Andrea Pellagatti1,2, Helen Turley1, Jacqueline
Boultwood1,2 & Richard Callaghan1,3
SUPPLEMENTARY DATA
Figure S1
Western immuno-blot data for enzymes and transporters
Table S1
Conditions for measurement of enzyme activity – coupled enzyme assays
Table S2
Conditions for measurement of enzyme/transporter expression
Target Enzyme
Buffer System
Enzymes Added
Biochemical
Added
Reaction
Monitored
Start of reaction
Hexokinase
0.05M Tris.HCl 13mM
MgSO4
(pH 8.0)
2.4U/ml G6PDH
0.55mM ATP
0.22mM NADP
NADP reduction
TS homogenate of 0.1mg
total protein content
Glucose 6 phosphate
Dehydrogenase
0.055M Tris.HCl
3.3mM MgSO4
(pH7.8)
0.2mM NADP
2mM G-6-P
NADP reduction
TS homogenate of 0.05mg
total protein content
Phospho-fructokinase
50mM Tris.HCl
5mM MgSO4
5mM NH4SO4
(pH 7.6)
0. 5mM F-6-P,
1mM ATP
0.25mM NADH
NADH oxidation
TS homogenate of 0.1mg
total protein content
Glutamate
dehydrogenase
0.1M Triethanolamine
(pH7.3)
50mM CH3COONH4
0.1mM NADH
0.25mM EDTA
33.8mM KG
NADH oxidation
TS homogenate of 0.1mg
total protein content
Lactate
dehydrogenase
0.2M Tris.HCl (pH7.3)
0.22mM NADH, 4mM
Na-pyruvate
NADH oxidation
TS homogenate of 0.05mg
total protein content
Pyruvate
Kinase
0.05M Imidazole
0.12M KCl
62mM MgSO4
(pH7.6)
1.48mM ATP
0.22mM NADH
3mM PEP
NADH oxidation
TS homogenate of 0.05mg
total protein content
2.5U TPI
1.5U aldolase
3.6U αGPDH
0.2mg/ml LDH
Supplementary Table 1:
Conditions for measurement of enzyme activity and monitored reactions
Enzyme activities were measured using spectrophotometric assays in a total volume of 1ml. The reactions were monitored by the continuous
recording of the change in absorbance caused by the oxidation or reduction of the co-factors NADH or NADP. The table provides information
on the buffers, biochemical and enzymes used in the reactions. The addition to initiate each reaction is also described. Enzyme abbreviations
include: G6PDH – glucose-6-phosphate dehydrogenase, TPI – triose phosphate isomerase, αGPDH – alpha-glycerophosphate dehydrogenase,
LDH – lactate dehydrogenase.
Target Enzyme
TS
homog
Hexokinase
50μg
Glucose-6-Phosphate
Dehydrogenase
10μg
Phosphofructokinase P
10μg
Phosphofructokinase M
10μg
Phosphofructokinase L
30μg
PFKFB3
10μg
Glutamate
Dehydrogenase
10μg
Pyruvate Kinase
10μg
Lactate Dehydrogenase
10μg
GLUT1
10μg
no heat
GLUT3
10μg
no heat
ASCT2
10μg no
heat
MCT1
10μg
no heat
MCT4
10μg
no heat
HIF 1
10μg
c-Myc
10μg
Primary Antibody
conditions
Rabbit Hexokinase II mAb
1:1000(v/v)
5% milk in PBS-T
Rabbit G6PDH
1:1000(v/v)
5% milk in PBS-T
Rabbit PFK-P mAb
1:1000(v/v)
5% milk PBS-T
Mouse PFK-M mAb
1:1000(v/v)
5% milk PBS-T
Mouse PFK-L mAb
1:500(v/v)
5% milk PBS-T
Rabbit PFKFB3 pAb
1:1000(v/v)
5% BSA PBS-T
Goat GDH
1:10000(v/v
5% milk in PBS-T
HRP-conjugated PK
1:10000(v/v)
5% milk in PBS-T
HRP-conjugated
1:10000(v/v)
5% milk in PBS-T
GLUT1 rabbit pAb
1:2000(v/v)
5% milk in PBS-T
GLUT3
1:1000(v/v)
5% milk in PBS-T
ASCT2 (G11) rabbit pAb
1:1000(v/v)
5% BSA in PBS T
MCT1 (H-70) Rabbit pAb
1:1000(v/v)
5% milk in PBS-T
MCT4 (H-90) Rabbit pAb
1:1000(v/v)
5% milk in PBS-T
HIF-1α Monoclonal
mouse purified IgG1
clone 54 1:1000(v/v)
5% milk in PBS-T
cMyc mouse mAb (HRP)
1:1000 (v/v)
5% milk in PBS-T
Secondary antibody
conditions
Anti Rabbit-HRP
1:2000 (v/v) in PBS-T
Anti Rabbit –HRP
1:2000(v/v) in PBS-T
Anti Rabbit-HRP
1:2000(v/v) in PBS-T
Anti mouse-HRP
1:2000(v/v) in 5% milk
PBS-T
Anti mouse-HRP
1:2000(v/v) in 5% milk
PBS-T
Anti Rabbit-HRP
1:2000(v/v) in 5% BSA
PBS-T
Anti Goat-HRP
1:2000(v/v) in 5% milk in
PBS-T
N/A
N/A
Anti Rabbit-HRP
1:2000(v/v) in 5% BSA
PBS-T
Anti Rabbit-HRP
1:2000(v/v) in 5% BSA
PBS-T
Anti Rabbit-HRP
1:2000 (v/v) in 5% BSA
PBS-T
Anti Rabbit-HRP
1:2000 (v/v) in 5% BSA
PBS-T
Anti Rabbit-HRP
1:2000 (v/v) in 5% BSA
PBS-T
Anti mouse-HRP
1:2000(v/v) in 5% milk
PBS-T
N/A
Supplementary Table 2:
Conditions
for
measurement
of
enzyme/transporter
expression by western blotting
The table shows the amount of homogenate used in western blots and the type, dilution and
buffer conditions for primary and secondary (where required) antibodies. Loading control for
the lanes was achieved by adding a fixed and known amount of total protein in the
homogenates. The phrase “no heat” indicates that samples in Laemmli sample buffer should
not be heated prior to SDS-PAGE.
Figure S1
The expression of metabolism related proteins in tumour spheroids
Tumour spheroids with diameters in the range 200-400m were grown under conditions of
normoxia (fully proliferative), severe hypoxia and those that induced quiescence. Following
incubation, homogenates were generated for the spheroids as described in materials and
methods. Homogenates were subjected to SDS-PAGE and western transfer to nitrocellulose.
The panels above panel show a representative western immunoblot of spheroid lysate for
tumour spheroids grown under normoxia (TSN), hypoxia (TSH) and quiescence (TSQ).
Equivalent loading between samples was achieved by using 10µg total protein per lane. The
TSQ samples are not relevant to this manuscript and will be discussed in another. However,
the samples provide further evidence for the interplay between metabolic
enzymes/transporters and the local micro-environment.