Fig. 3

Inhibition of HNE-induced WNT signalling by NAC. a ARPE19 cells were transfected with TOPFLASH or FOPFLASH plasmid for 8 h and then pretreated for 1 h with NAC (1 mmol/l) prior to challenge with HNE (10 μmol/l) for another 16 h. TOPFLASH activity (white bars) and FOPFLASH activity (grey bars) were measured by luminometer and normalised to Renilla luciferase activity. Values mean ± SD; n = 3; *p < 0.05 for HNE compared with control; ^† p < 0.05 for HNE+NAC compared with HNE. b ARPE19 cells were pretreated for 1 h with NAC (1 mmol/l) prior to challenge with HNE (10 μmol/l) for 6 h. Phosphorylated (p)LRP6 and total LRP6 levels were determined by western blot analysis using 50 μg of total proteins from each sample. The blot is representative of three independent experiments. c pLRP6 and (d) total LRP6 levels were quantified by densitometry and normalised to β-actin levels. Values are per cent of those in control cells, expressed as mean ± SD; n = 3; **p < 0.01 for HNE compared with control; ^† p < 0.05 and ^†† p < 0.01 for HNE+NAC compared with HNE. e ARPE19 cells were pretreated for 1 h with NAC (1 mmol/l) prior to challenge with HNE (10 μmol/l) for 16 h, followed by addition of cycloheximide (50 μg/ml) to block protein translation. Cells were collected at 0, 3, 5, 7 and 9 h following the addition of cycloheximide (Chx0). Total LRP6 protein levels at each time point were semi-quantified using western blot analysis, normalised to β-actin levels and are expressed (f) as per cent of the LRP6 levels prior to addition of cycloheximide. Blots (e) are representative of three independent experiments. Values (f) are mean ± SD; n = 3. Diamonds, control; squares, HNE; triangles, HNE + NAC