Co-transfection of the DAF promoter construct and the Sp1 vector in HT1080 cells does not alter basal activity. The average fold change of luciferase activity from cells co-transfected with empty vector (pCMV) as control relative to those transfected with Sp1 transcription factor (pCMV-Sp1), in addition to either WT or mut DAF promoter, is similar. Mean data (±s.d.) of duplicate experiments.

The c.-198C>G (mutant) DAF SNP is regulatory. (a) Transfection of the mutant DAF regulatory construct activates DAF promoter activity (as measured by luciferase assay) compared with transfection with WT promoter construct in COS-7 and HT1080 cells (normalized to 1); the plasmid pRL-TK (50 ng) containing the thymidine kinase promoter driving the expression of a Renilla reporter was used as an internal control for transfection efficiency and firefly luciferase activity was normalized to Renilla luciferase activity. Results representative of two independent experiments carried out in duplicate. (b) Luciferase assay in C2C12 muscle cells transfected with the mutant DAF promoter compared with WT controls (normalized to 1) shows activation at 0–2 days of differentiation. Mean data (±s.d.) of duplicate experiments. Undiff, undifferentiated; 2dd, 2 days in differentiation; and 10dd, 10 days in differentiation medium. (c) Quantitative RT-PCR of gDAF mRNA extracted from lymphoblastoid cell lines from three patients with the c.-198C>G (MG1 to MG3), one MG patient without the SNP (WT-MG) and two controls (WT1 and WT2). Absolute mRNA expression levels (normalized to GUS1B) are extrapolated from a standard curve. Data (mean±s.d.) from two independent experiments carried out in duplicate and expressed as fold change over non-LPS controls.

Effect of LPS treatment on gDAF RNA and protein expression in lymphoblasts. (a) Total RNA was extracted from lymphoblast cell lines from three controls (WT1 and WT2 and one MG patient without the SNP, WT-MG) and four MG patients with the c.-198C>G SNP (MG1 to MG4), with and without LPS stimulation (10 μg/ml) for 6 h. Relative expression (normalized to GUS1B) by qRT-PCR is shown expressed as fold change over non-LPS control for each cell line (normalized to 1). Data (mean±s.d.) from triplicate experiments. (b) Western blot analysis of DAF protein levels in control (WT1) and MG patient (MG1) derived cell lines. The cells were treated with 10 μg ml⁻¹ of LPS for 6 h. Protein was extracted under non-reducing conditions and quantified using BCA standard curve method (Materials and methods). For each sample, 30 μg of protein was loaded on an SDS-PAGE gel and p38 was used as a loading control to ensure equal loading of protein. DAF protein levels increased in control cell line but decreased in the MG patient cell line on treatment with LPS. (c) The bar graph represents quantitative DAF protein results by showing densitometry readings from the autoradiograph of DAF protein levels normalized to p38 protein levels. For the control cell line, DAF protein levels showed an increase of approximately two-folds as a result of LPS treatment. On the contrary, the MG SNP cell line showed a decrease of approximately two-fold when treated with LPS.