Supplementary Information for Yee, Chen and Giacomini Supplementary Information Correspondence Letter Sook Wah Yee*, Ligong Chen*, Kathleen M. Giacomini # * These authors contributed equally in this correspondence letter. # Correspondence should be addressed to: [email protected] Nature Genetics: doi:10.1038/ng.2236
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Supplementary Information for Yee, Chen and Giacomini
Supplementary Information Correspondence Letter Sook Wah Yee*, Ligong Chen*, Kathleen M. Giacomini# * These authors contributed equally in this correspondence letter.
# Correspondence should be addressed to: [email protected]
Nature Genetics: doi:10.1038/ng.2236
Supplementary Information for Yee, Chen and Giacomini
Supplementary Figures Supplementary Figure 1. Metformin activation of AMP-activated protein kinase (AMPK) in H4IIE cells. H4IIE cells were pre-treated with or without, cimetidine (1.0 mM), KU-55933 (10 µM), imatinib (10 µM) and verapamil (200 µM) for 1 hour and then for 3 hours with or without metformin following the methods described by Zhou et al.1. Results from (a) Western blot assays and (b) densitometry assays were obtained following methods described previously2-4. The bar chart from the densitometry study (b) represents the intensity of each band (P-AMPK) normalized to the total AMPK (T-AMPK) based on 3 independent experiments. Metformin (2.5 mmol/L) stimulated AMPK phosphorylation and the inhibitors, cimetidine (1.0 mM), KU-55933 (10 µM), imatinib (10 µM) and verapamil (200 µM) inhibited metformin action on AMPK phosphorylation. Data are expressed as mean ± standard deviation from three independent Western blots.
Nature Genetics: doi:10.1038/ng.2236
Supplementary Information for Yee, Chen and Giacomini
Supplementary Figure 2. Time course of effect of KU-55933 on metformin uptake in H4IIE cells. The cells were pre-treated with DMSO or with KU-55933 for 30 min and then 2.5 mM metformin (plus 10 µM [14C]-metformin) for 1, 5, 10, 20, 30, 45, 60 and 120 min at 37 °C. Amount of [14C]-metformin in cells was then measured. Results are the mean ± standard deviation for metformin uptake from triplicate wells (nmol/mg protein). KU-55933 (10 µM) inhibited metformin uptake (*** P<0.001, ** P<0.01, * P<0.05, paired student t-test) compared with cells pre-incubated with DMSO alone.
Nature Genetics: doi:10.1038/ng.2236
Supplementary Information for Yee, Chen and Giacomini
Supplementary Figure 3. Effect of KU-55933 on metformin uptake in Atm +/+ (A29) and Atm -/- (A38) cells transiently expressing mouse Oct1 or expression vector (EV), pcDNA5FRT. (a) – (c) A29 and A38 cells5 were seeded at 1 x 105 cells in 12-well plate and then next day transfected with 1.6 µg expression vector (mouse Oct1 or EV) per well plus 4 µL of Lipofectamine 2000 (Invitrogen). 30 hours after transfection, the cells were washed twice with Hank’s balanced salt solution before pre-treated with DMSO or with 10 µM KU-55933 for 30 min. Transfection efficiencies of mouse Oct1 were similar between the two cell lines using qRT-PCR (data not shown). Metformin uptake was performed at 37 °C for 10 min and 30 min with 40 µM unlabeled metformin (plus 10 µM [14C]-metformin). Results are the mean ± standard deviation for metformin uptake from triplicate wells. A29 and A38 cells expressing mOCT1 showed significantly greater metformin uptake compared to cells transfected with vector pcDNA5FRT (EV) only. KU-55933 (10 µM) inhibited metformin uptake in both A29 and A38 cells expressing mOCT1 compared with cells pre-incubated with DMSO alone. P values were calculated using Student t-test. aP < 0.001, bP <0.01, cP<0.05 vs cells transfected with mouse Oct1 and pre-incubated with DMSO.
References for supplementary figures 1. The GoDARTS and UKPDS Diabetes Pharmacogenetics Study Group, Zhou K, Bellenguez C,
Spencer CC, Bennett AJ, Coleman RL, et al. Nat. Genet. 43,117-120 (2011). 2. Shu Y, Sheardown SA, Brown C, Owen RP, Zhang S, Castro RA, et al. J. Clin. Invest. 117,1422-
1431 (2007). 3. Ahlin G, Chen L, Lazorova L, Chen Y, Ianculescu AG, Davis RL, et al. Pharmacogenomics J. Jun
22 (2010). 4. Chen L, Pawlikowski B, Schlessinger A, More SS, Stryke D, Johns SJ, et al. Pharmacogenet.
Genomics 20, 687-699 (2010). 5. Yang DQ and Kastan MB. Nat. Cell Biol. 2, 893-898 (2000).
Nature Genetics: doi:10.1038/ng.2236
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