Table 1. The effects of MAN2C1- and ENGASE-targeted siRNA duplexes on fOS steady state levels in HepG2 cells.
| ENG-3 | M2C1-1 | |||||
| Component (% in MBCc) | Fold Ctrl a (% in MBC b) | |||||
| fOSGN2d | G1M9 | 11.1 | -e | |||
| M9 | 6.1 | - | ||||
| M8 | 7.0 | - | ||||
| fOSGN | G1M9 | 1.2 | 6.3 | (4.2) | ||
| M9 | (7.4) | 1.2 | 6.6 | (6.3) | ||
| M8 | (12.4) | 1.0 | 5.1 | (8.6) | ||
| M7 | (10.4) | 0.9 | 1.2 | (10.2) | ||
| M6 | (13.9) | 0.5 | (9.2) | 0.4 | (12.0) | |
| M5 | (12.6) | 0.4 | (10.7) | 0.1 | (5.7) | |
| M4 | (53.0) | 0.4 | (48.9) | 0.1 | (60.2) | |
| M3 | (>90) | 0.4 | (>90) | 0.2 | (>90) | |
HepG2 cells were transfected with control and ENGASE (ENG-3)- and MAN2C1 (M2C1-1)-targetted RNAi duplexes prior to SLO permeabilisation to generate cytosol and MBC fractions. After extraction, purification, derivatisation, fOS were resolved by HPLC as described for Fig 3A and B.
Where peaks were clearly identified, the distribution of each component in the cytosol and MBC fractions after permeabilisation with SLO was calculated as % total component in MBC (% in MBC). Where this value is not shown, low levels of material recovered from the MBC fraction did not permit unambiguous detection of component.
The calculations described aboveb were also performed for fOS identified in cells transfected with the control siRNA duplex.
fOSGN2 were quantitated by HPLC and fluorescence detection of total fOS-AP before and after endoH treatment as described in Fig 3A. HPLC profiles indicate the presence of at least two components migrating similarly to Man8GlcNAc2-AP (Fig 3A, right panel) and these two components were quantitated together. Small amounts of a component migrating as Man7GlcNAc2-AP (Fig 3A, right panel) were also identified but were not quantitated.
Transfection of HepG2 cells with M2C1-1 provoked large accumulations of fOSGN which masked the appearance of fOSGN2.