Supplementary information
Crucial involvement of xanthine oxidase in the intracellular signalling networks
associated with human myeloid cell function
Maryam Abooali, Gurprit S. Lall, Karen Coughlan, Harjinder S. Lall, Bernhard F. Gibbs* &
Vadim V. Sumbayev*
School of Pharmacy, University of Kent, Chatham Maritime, ME4 4TB, United Kingdom
Materials
RPMI-1640 medium, foetal calf serum and supplements, DOTAP transfection reagent,
purified bovine milk xanthine oxidase and pharmacological inhibitors were purchased from
Sigma (Suffolk, UK). The following products were used: allopurinol (A8003), sodium
tungstate (T2629), LY294002 (L9908), SB203580 (S8307), rapamycin (37094). The AP-1
inhibitor SR11302 (2476) was from Tocris Bioscience (Bristol, UK). Maxisorp™ microtitre
plates were obtained from Nunc (Roskilde, Denmark). Antibodies to XOD, mTOR, β-actin,
phospho-S2448 mTOR and phospho-T2446 mTOR were from Abcam (Cambridge, UK).
Goat anti-mouse and goat anti-rabbit fluorescently-labelled antibodies were obtained from LiCor (Lincoln, Nebraska USA). Human recombinant SCF was obtained from Invitrogen
(Paisley, UK). All other chemicals were of the highest grade of purity commercially
available.
Cells
THP-1 human leukaemia monocytic macrophages were purchased from the European
collection of Cell Cultures (Salisbury, UK). These cells, as well as MCF-7 breast cancer cells,
were grown in RPMI 1640 media supplemented with 10% foetal calf serum, penicillin (50
IU/ml) and streptomycin sulphate (50 µg/ml). Primary human basophils were purified from
buffy coats (purchased from the National Health (NHS) Blood and Transfusion Service
following ethical approval; REC reference 12/WM/0319) as previously described9. LAD2
mast cells were kindly provided by A. Kirshenbaum and D. Metcalfe (NIH, USA)24. Cells
were cultured in Stem-Pro-34 serum-free media in the presence of 100 ng/ml SCF. For
analysis we used whole cell extracts which were obtained using lysis buffer (pH 7.5)
containing 50 mM tris, 150 mM NaCl, 5 mM EDTA and 1 % Nonidet-P-40 (NP-40). 1 mM
phenylmethanesulfonyl fluoride (PMSF) was supplied just before use.
Mice
Six week old CD1 male mice (25 ± 2.5 g) were used for the experiments following approval
by the Institutional Animal Committee and handled in accordance with the Animals
(Scientific Procedures) Act 1986 and Helsinki Declaration. Blood serum and cellular
fractions (peripheral blood mononuclear cells) were isolated as described before7. Livers
were also isolated and homogenised in cell lysis buffer at aratio of 1:10. Each group of
animals received two intraperitoneal injections: 1) two independent injections of 0.9% NaCl
(control); 2) one injection of 1 mg/kg PGN and another of 0.9% NaCl. After 4 h animals were
euthanized and blood and liver were collected. Blood cells were lysed using the same lysis
buffer as described above (see “Cells” section). This buffer was also used for preparing liver
homogenates (at a ratio of 1 : 20).
Supplementary figure 1. Differential expression of xanthine oxidase in different cell
types and mouse liver. A full range membrane.
For more details please see the legend to Figure 1. The full length XOD as normally comes
slightly above 150 KD. Large XOD subunit is also often visible (between 70 and 100 KD)
upon loading of high protein amounts as well as high levels of protein XOD expression.
XOD
Actin
Supplementary figure 2. A full range membrane of the Western blot presented in Figure
3A.
Approximately 40-50 μg protein was loaded followed by the analysis outlined in the main
text and Supplementary Figure 1.
XOD
Actin
Supplementary figure 3. A full range membrane of the Western blot presented in Figure
3B.
Approximately 90 μg protein was loaded (to be able to visualise XOD in all samples, since
the AP-1 inhibitor causes a strong downregulation in XOD protein levels) followed by the
analysis outlined in the main text and Supplementary Figure 1. Because of high protein
loading, the blot was stripped following XOD staining (using ReBlot™ Plus Kit from
Chemicon International, CHEMICON Europe, Ltd., Chandlers Ford, Hampshire, UK) and
reprobed with anti-actin antibody.
Supplementary figure 4. HIF-1α mRNA levels in normal and HIF-1α knockdown THP1 cells. For more details please see the legend to the Figure 3.
phospho-p38
p38
Supplementary figure 5. A full range membrane of the Western blot presented in Figure
4A.
Approximately 50 μg protein was loaded followed by the analysis outlined in the main text
and Supplementary Figure 1. Following the transfer of proteins from the gel onto the
nitrocelllose membrane, the membrane was cut above 50 KD in order to reduce consumption
of anti-p38 and anti-phospho-p38 antibodies.
Supplementary figure 6. Characterisation of XOD inhibitors and activators. Kinetics of
the effects of different compounds were analysed. Allopurinol, rapamycin (A), sodium
tungstate and ammonium molybdate (B) had a direct effect on the activity of purified bovine
milk XOD. Other compounds tested (Supplementary table 1) had no effect on XOD activity
(n=6). Results are presented as Lineweaver–Burk plots based on the equation:
1/v = 1/[S] x Km/Vmax + 1/Vmax.
Ki and Ka values were calculated based on the Lineweaver-Burk plot:
Isosteric inhibition: Ki = Kmi [I] / (Kmi – Km)
Non-competitive inhibition: Ki = Vmax [I] / (Vmax – Vmax i)
Activation by Mo: Ka = Vmax [A] / Vmax a - Vmax
Supplementary figure 7. Inhibition of XOD provokes increases in intracellular AMP
levels as well as inhibitory (T2446) mTOR phosphorylation. (A) THP-1 cells were
exposed to 1 μg/ml LPS, 1 μg/ml PGN, 0.1 μg/ml R848 as well as 0.1 μg/ml SCF for 4 h with
or without 1 h pre-treatment with 250 μg/ml allopurinol. Phospho-S2448 mTOR and
phospho-T2446 mTOR were measured by ELISA. (B) THP-1 cells were exposed to 1 μg/ml
LPS, 1 μg/ml PGN, 0.1 μg/ml R848 as well as 0.1 μg/ml SCF for 4 h with or without 1 h pretreatment with 100 μg/ml sodium tungstate. XOD activity, intracellular AMP levels and
phospho-T2446 were measured. Results are shown as mean values ± S.D. of at least three
independent experiments; * – p < 0.01 vs. control; a - p < 0.01 vs. corresponding treatment.
Supplementary figure 8. Molybden-induced XOD activity does not affect mTOR
phosphorylation. THP-1 cells were exposed to 100 μg/ml ammonium molybdate followed
by detection of XOD activity, intracellular AMP levels as well as phospho-S2448/phosphoT2446 mTOR. Results are shown as mean values ± S.D. of at least three independent
experiments; (n=3) * – p < 0.01 vs. control; a - p < 0.01 vs. corresponding treatment.
Supplementary figure 9. Overview of the involvement of XOD in the biological
responses of human myeloid cells. The ASK1 pathway is shown as one of the possible p38
MAP kinase activators during the biological responses of human myeloid cells21. 1 and 6 –
5’-nucleotidase, 2 and 7 – nucleoside phosphorylase, 3 – AMP deaminase, 4 – Adenosine
deaminase, 5 – adenase, 8 – hypoxanthine-guanine phosphoribosyl transferase (HGPRT), 9 –
adenylosuccinate synthase, 10 – adenylosuccinate liase.
Supplementary table 1. Characterisation of the kinetics of different compounds on XOD
activity
Agent
Allopurinol
Na2WO4
(NH4)6Mo7O24
LY294002
SB203580
CoCl2
Rapamycin
Concentration
0.5 mM
100 μg W/ml
100 μg Mo/ml
50 μM
10 μM
100 μM
20 μM
Effect on XOD activity
Inhibitor (competitive)
Inhibitor (non-competitive)
Activator
No effect
No effect
No effect
Activator
Ki or Ka
1.82 mM
1.09 mM
0.6 mM
N/A
N/A
N/A
2.36 μM