Fig. 4.

The effect of hypoxia on the assembly of the heteromeric glycosyltransferase complexes in COS-7 cells. a) Heteromeric N-glycosyltransferase complexes. b) Heteromeric O-glycosyltransferase complexes. In both cases, cells were transfected with the indicated mVenus and mCherry enzyme constructs. The enzyme pairs were chosen based upon their previously established interactions (Hassinen et al., 2011). Thereafter, cells were cultivated in both normoxic and hypoxic conditions for 24 h before quantification of the FRET signals with the high content imaging system. FRET efficiencies were calculated as described in the “Materials and Methods” section, see the Supplementary Appendix) and expressed as percentages (mean ± SD, n = 3, 15,000 cells each)) from the normoxic (control) values (set to 100%). Only statistically significant changes from the normoxic control cells are marked with stars (p < 0.05*, p < 0.01**, p < 0.001***). c. Time dependent changes on the assembly of the depicted enzyme complexes by moderate hypoxia. Cells were grown in hypoxic conditions (5% O₂) and imaged at the indicated times (0, 4, 8, 24 h) before determining the FRET efficiencies for each enzyme pair. The data are expressed as percentages (mean ± SD, n = 3, 15,000 each) from the control (0-time point) values. Statistically significant changes are marked with stars (p < 0.05*, p < 0.01**, p < 0.001***). d) Sensitivity of the enzyme complexes to reducing agent (DTT). Cells were processed as above except that they were grown in the presence of different concentrations of DTT for 20 h before quantification of the FRET efficiencies. The data are expressed as percentages (mean % + SD, n = 3 (15,000 each) from the control (no DTT) values. Statistically significant changes are marked with stars (p < 0.05*, p < 0.01**, p < 0.001***).