Figure 2

LRRK2 promotes httQ74 toxicity and accumulation of endogenous proteins. (a and b) COS-7 cells were co-transfected with the aggregate-prone protein httQ74-EGFP along with LRRK2 (Flag) or httQ23. Cells were fixed 48 h after transfection and stained with an antibody against Flag and the nuclear dye DAPI. (a) Aggregation of httQ74-EGFP and (b) cell death were scored in 200 cells per replicate. Graphs show means±S.E.M. of three independent experiments performed in triplicates. Statistical analysis was performed by unconditional logistical regression analysis. ^***P<0.001, N=3. (c) HeLa cells were transiently co-transfected with GFP along with either empty vector, httQ23, WTLRRK2 or GSLRRK2. After 48 h of transfection, cellular GFP fluorescence was measured by flow cytometry. Left: representative flow cytometry histogram shows that GFP fluorescence was increased in HeLa cells that expressed WTLRRK2 or GSLRRK2 (dark grey and light grey line, respectively), compared with cells transfected with vector or httQ23 (black and thin black line, respectively). Right: quantification of cellular fluorescence of three independent experiments performed in triplicates. The geometric mean of cellular fluorescence was normalised to vector-transfected cells within each experiment, error bars represent standard deviation of all replicates. Unpaired, two-tailed Student's t-test was performed on raw replicate data from each individual experiment and representative P-values are shown. ns=not significant, ^***P<0.005, N=3. (d) HeLa cells were transiently co-transfected as in (c). Additionally, the effect of LRRK1 overexpression was tested on levels of GFP. Overexpression of LRRK2 but not LRRK1 or httQ23 lead to increased fluorescence, compared with vector-transfected cells. Graph represents data as above from three independent experiments performed in triplicates (N=3). (e) The human neuroblastoma cell line SHSY5Y was transfected and analysed as HeLa cells in (d). Increased GFP fluorescence was measured in cells transfected with LRRK2, but not in httQ23- or LRRK1-transfected cells. Graph represents data as above from four independent experiments performed in triplicates (N=4). (f) HeLa cells were transfected with GFP and DsRed and either control DNA or LRRK2. After 48 h of transfection cells were sorted for GFP and DsRed double-positives and grown for 24 h before cell collection. An additional vector control was treated for 12 h with MG132, a proteasome inhibitor, before collection. p53 levels were analysed by western blotting. Left: blots represent one of seven independent experiments. Densitometry results beneath the blots are normalised to actin. Right: quantification of seven independent experiments. Data are normalised to vector-transfected cells and graphs show mean±S.D. Raw data were analysed by two-tailed, paired Student's t-test. ^*P< 0.05, ^**P<0.01, N=7. Note, that MG132 treatment was only performed in two of the seven experiments