Fig. S1 Biotinylated GLUT7-GFP Protein Expression in Xenopuslaevis Oocytes

Supplementary material

Fig. S1 Biotinylated GLUT7-GFP protein expression in Xenopuslaevis oocytes

Western blot of oocyte protein stained with GFP (red) and actin (green) antibodies. Lane 1: NI, no Biotin incubation, total lysate; Lane 2: NI, no Biotin incubation, streptavidin-pull down; Lane 3: NI, incubated with Biotin, total lysate; Lane 4: NI, incubated with Biotin, streptavidin-pull down; Lane 5: Marker; Lane 6: GLUT7-GFP, no Biotin incubation, total lysate; Lane 7: GLUT7-GFP, no Biotin incubation, streptavidin-pull down; Lane 8: GLUT7-GFP, incubated with Biotin, total lysate; Lane 9: GLUT7-GFP, incubated with Biotin, streptavidin-pull down.

Fig. S2 Fructose uptake into Xenopuslaevis oocytes expressing GLUT7

GLUT7 injected oocytes were incubated for 5 minutes with fructose (200 µM) 4 days after injection of 20 ng cRNA. Non-injected (NI) oocytes were treated equally. Bars represent mean values of 8 oocytes. Error bars indicate the standard deviation. Mann-Whitney-U-test was used to test for significant differences compared to NI oocytes.

Fig. S3Glucose uptake into Xenopuslaevis oocytes expressing GLUT7

GLUT7 injected oocytes were incubated for 5 minutes with glucose (1 mM) 4 days after injection of 23 ng cRNA. Non-injected (NI) oocytes were treated equally. Bars represent mean values of 10 oocytes. Error bars indicate the standard deviation. T-test was used to test for significant differences compared to NI oocytes.

Fig. S4 Fructose uptake into Xenopuslaevis oocytes expressing GLUT7 (20-200 µM)

GLUT7 injected oocytes were incubated for 30 minutes with fructose (20, 50, 100 or 200 µM) 4 days after injection of 23 ng cRNA. Non-injected (NI) oocytes were treated equally. Bars represent mean values of 10 oocytes. Error bars indicate the standard deviation. T-test was used to test for significant differences compared to NI oocytes.

Fig. S5 Fructose uptake into Xenopuslaevis oocytes expressing GLUT7 (3,4 or 5 days)

GLUT7 injected oocytes were incubated for 2 minutes with fructose (200 µM) 3, 4 or 5 days after injection of 23 ng cRNA. Non-injected (NI) oocytes were treated equally. Bars represent mean values of 8-10 oocytes. Error bars indicate the standard deviation. Mann-Whitney-U-test was used to test for significant differences compared to NI oocytes (*** p<0.001).

Fig. S6 Fructose uptake into Xenopuslaevis oocytes expressing GLUT7 or GLUT7-HA

a) GLUT7 and GLUT7-HA (with HA-tag) injected oocytes were incubated for 5 minutes with fructose (1 mM) 4 days after injection of 19 or 32 ng cRNA. Non-injected (NI) oocytes were treated equally. Bars represent mean values of 7-10 oocytes. Error bars indicate the standard deviation. T-test was used to test for significant differences compared to NI oocytes.

b) Western blot of oocyte protein extracts (10 µg per lane) stained with HA (red) and actin (green) antibodies. Lane 1: GLUT7-HA 32 ng, Lane 2: GLUT7-HA 19 ng.

Fig. S7 Fructose uptake into Xenopuslaevis oocytes expressing GLUT7 (fasted)

GLUT7 injected oocytes were incubated for 5 minutes with fructose (1 mM) 4 days after injection of 19 or 32 ng cRNA. Non-injected (NI) oocytes were treated equally. Some oocytes were fasted for 10 minutes in Barth’s solution without pyruvate prior to incubation. Bars represent mean values of 9-10 oocytes. Error bars indicate the standard deviation. T-test was used to test for significant differences compared to NI oocytes.

Fig. S8 Fructose uptake into HuH7 cells expressing GLUT7

HuH7 cells were transfected with 10 µg pCEP4 (vector control), pCEP4-GLUT7 or pCEP4-GLUT7-HA and 20 µl Turbofect.

a) Vector control, GLUT7 and GLUT7-HA transfected cells were incubated for 5 minutes with 1 mM fructose. Bars represent mean values of 2 wells (vector control, GLUT7) or value of 1 well (GLUT7-HA).

b) Western blot of HuH7 (GLUT7-HA transfected) protein extracts (10 µg per lane) stained with HA (red) and actin (green) antibodies. Lane 1: HA, Lane 2: Actin.

Fig. S9 Glucoseuptake into HuH7 cells expressing GLUT7

HuH7 cells were transfected with 10 µg pCEP4 (vector control), pCEP4-GLUT7 or pCEP4-GLUT7-HA and 20 µl Turbofect.

a) Vector control, GLUT7 and GLUT7-HA transfected cells were incubated for 5 minutes with 1 mM glucose. Bars represent mean values of 2 wells (vector control, GLUT7) or value of 1 well (GLUT7-HA).

Fig. S10 Fructose uptake into CHO cells expressing GLUT7

CHO cells were transfected with 6 µg pCEP4 (vector control), pCEP4-GLUT7, pCEP-GLUT7-HA or pCEP4-GLUT7-GFP and 12 µl FuGENE HD in 6 well plates.

a) Vector control, GLUT7 and GLUT7-GFP transfected cells were incubated for 5 minutes with 1 mM fructose. Bars represent mean values of 4 wells (vector control, GLUT7) or value of 2 wells (GLUT7-GFP). Error bars indicate the standard deviation.

b) Western blot of CHO protein extracts (10 µg per lane) stained with HA, GFP (red) and actin (green) antibodies. Lane 1: GLUT7-HA, Lane 2: GLUT7-GFP.

Fig. S11 Fructose and glucoseuptake into CHO cells expressing GLUT7

CHO cells were transfected with 1 µg pCEP4 (vector control), pCEP4-GLUT7, pCEP-GLUT7-HA or pCEP4-GLUT7-GFP and 2 µl FuGENE HD in 24 well plates.

a) Vector control, GLUT7, GLUT7-HA and GLUT7-GFP transfected cells were incubated for 1 minute with 1 mM fructose or glucose. Bars represent mean values of 8-9 wells (vector control, GLUT7) or value of 1 well (GLUT7-HA, GLUT7-GFP). Error bars indicate the standard deviation. T-test was used to test for significant differences compared to control cells.

Fig. S12GLUT5-GFP p.I296V protein expression in Xenopuslaevis oocytes and NIH-3T3 cells

a) Oocytes were embedded in paraffin and cut into slices. GFP fluorescence was visualized using a Leica microscope (20x magnification). b) Western blot of oocyte protein extracts (10 µg per lane) stained with GFP (red) and actin (green) antibodies. Lane 1: NI, Lane 2: GLUT5-GFP, Lane 3: GLUT5-GFP p.I296V. c) Stable cell lines were created using retroviral transduction. GFP fluorescence of GLUT5-GFP and GLUT5-GFP p.I296V expressing cells was visualized using a Leica microscope (10x magnification). d) Western blot of NIH-3T3 protein extracts (5 µg per lane) stained with GFP (red) and actin (green) antibodies. Lane 1: GFP, Lane 2: GLUT5-GFP, Lane 3: GLUT5-GFP p.I296V.

Fig. S13 Fructose, glucose and galactose uptake into GLUT2 and GLUT2-GFP expressing Xenopuslaevis oocytes

GLUT2 and GLUT2-GFP injected oocytes were incubated for 10 minutes with fructose, glucose or galactose (1 mM) 4 days after injection of 13.8 ng cRNA. Non-injected (NI) oocytes were treated equally. Bars represent mean values of 9-10 oocytes. Error bars indicate the standard deviation. Mann-Whitney-U test was used to test for significant differences compared to NI oocytes (***p<0.001).

Fig. S14 GLUT2-GFP p.I322V protein expression inXenopuslaevis oocytes

Fig. S15GLUT9a-GFP and GLUT9b-GFP protein expression in Xenopuslaevis oocytes

Fig. S16 Fructose uptake into Xenopuslaevis oocytes expressing GLUT9

GLUT9a, GLUT9b, GLUT9a-GFP, GLUT9b-GFP and GLUT5-GFP injected oocytes were incubated for 30 minutes with fructose (1 mM) 4 days after injection of 13.8 ng cRNA. Non-injected (NI) oocytes were treated equally. Bars represent mean values of 8-10 oocytes. Error bars indicate the standard deviation. Mann-Whitney-U-test was used to test for significant differences compared to NI oocytes (*** p<0.001).

Relative abundance
Vector control / GLUT7 / GLUT7-GFP
Substance / Mean / SD / Mean / SD / Mean / SD
Arabinose / 7174359 / 1633067 / 9331271 / 3674324 / 6370534 / 2313918
Fucose / 5160864 / 1496875 / 7183201 / 3110669 / 4772705 / 1687472
Galactose / 27231571 / 5368427 / 38999483 / 11073002 / 31034139 / 10441008
Myo-Inositol / 93176226 / 8939917 / 95782501 / 16442642 / 93960190 / 18550337
Lactose / 3344708 / 938639 / 5013920 / 1958739 / 3393015 / 1047898
Maltose / 2430093 / 634900 / 3503342 / 1209004 / 2361079 / 605046
Mannitol / 94776 / 16985 / 138405 / 55081 / 142623 / 49162
Mannose / 24641190 / 5207615 / 32025472 / 9293606 / 26022348 / 8882897
Palatinose / 4204709 / 1185909 / 6546572 / 2510334 / 4228790 / 1123300
Raffinose / 2387874 / 649003 / 4018268 / 1548521 / 2417842 / 645964
Rhamnose / 2687766 / 729123 / 2767867 / 2411506 / 2464458 / 881015
Sorbitol / 189387 / 157541 / 154527 / 140026 / 139596 / 139099
Sucrose / 10774629 / 2446756 / 14880345 / 4744808 / 10759748 / 2727313
Xylose / 2878064 / 475399 / 3727785 / 1052708 / 3226247 / 1093072
GFP control / GLUT5-GFP
Mean / SD / Mean / SD
Fructose / 10578182 / 3085808 / 25671980 / 6408830

Table S1 Relative uptake of substances into NIH-3T3 cells expressing GLUT7 and GLUT7-GFP

GLUT7, GLUT7-GFP and vector control cells were incubated for 10 minutes with a mixture of D-arabinose, L-fucose, D-galactose, D-lactose, D-maltose, D-mannose, D-mannit, myo-inositol, palatinose, D-raffinose, L-rhamnose, D-ribose, D-sorbitol, D-sucrose and D-xylose (1g/l each). GLUT5-GFP and GFP control cells were incubated with the same mixture containing additionally 1 g/l D-fructose. The relative abundance was measured using GC-MS. Mean values and standard deviations (SD) from 3-4 wells are shown.