Western blot analysis of LRP expression in MDA-MB 231 cells 24 h and 48 h post-transfection with siRNA-LAMR1.

<p>No significant decrease in expression of the 37-kDa LRP was observed 24 h and 48 h after transfection when compared to non-transfected cells. The standard deviation is represented by error bars (n = 3).</p

Cell viability of MDA-MB 231 cells 24 h and 48 h after downregulation of LRP.

<p>MDA-MB 231 cells were transfected with siRNA-LAMR1 and cell viability was determined with the MTT assay. PCA (8 mM) and siRNA-scr were used as positive and negative controls, respectively. The viability was compared to the untreated control set at 100%. Error bars indicate standard deviation (n = 3).</p

The effect of siRNA-mediated downregulation of LRP expression on nuclear morphology.

<p>72 h post-transfection, MCF-7, MDA-MB 231 and WHCO1 cells were stained with Hoescht 33324 and viewed by immunofluorescence microscopy. siRNA-LAMR1 treated MCF-7, MDA-MB 231 and WHCO1 cells displayed condensed nuclei and decreased nuclear integrity (indicated by white arrows), compared to untreated and siRNA-scr treated cells (8mM PCA was used as a positive control).</p

The effect of siRNA-mediated downregulation of LRP expression on cell membrane integrity.

<p>72 h post-transfection, MCF-7, MDA-MB 231 and WHCO1 cells were stained with Annexin-V FITC and 7-AAD then analysed by flow cytometry. siRNA-LAMR1 treated MCF-7, MDA-MB 231 and WHCO1 cells revealed high FITC and 7-AAD signals indicative of cells undergoing early and late apoptosis, compared to untreated and siRNA-scr treated cells (8mM PCA was used as a positive control).</p

Sequence of control siRNA-RLUC as well as siRNA used for downregulation of LRP/LR.

<p>Sequence of control siRNA-RLUC as well as siRNA used for downregulation of LRP/LR.</p

Changes in nuclear area after siRNA-mediated downregulation of LRP.

<p>72 h post-transfection, MCF-7, MDA-MB 231 and WHCO1 cells were stained with Hoescht 33324 and viewed by immunofluorescence microscopy. The change in nuclear morphology was quantified with ImageJ software and is represented as the average nuclear area. The error bars indicate the standard deviation and a significant difference (* p < 0.05, ** p < 0.01, *** p <0.001) between the untreated control and the treated samples is shown by an asterisk.</p

The effect of siRNA-mediated downregulation of LRP expression on the cellular viability of WHCO1, MCF-7 and MDA-MB 231 cells.

<p>The viability of WHCO1, MCF-7 and MDA-MB 231 cells was analysed 72 h post-transfection using an MTT assay. siRNA-LAMR1 treated WHCO1, MCF-7 and MDA-MB 231 as well as esiRNA-RPSA treated MDA-MB 231 cells revealed a significant reduction in cellular viability compared to untreated cells set to 100%. 8mM PCA and siRNA-scr were used as positive and negative controls, respectively. The error bars represent the standard deviation (n = 3) and a significant difference (* p < 0.05, ** p < 0.01, *** p <0.001) between the untreated control and the treated samples is indicated by an asterisk.</p

LRP expression in WHCO1, MDA-MB 231 and MCF-7 cells 72 h post-transfection.

<p>WHCO1 and MCF-7 cells were transfected with siRNA-LAMR1 and MDA-MB 231 cells with siRNA-LAMR1 and esiRNA RPSA. Densitometric analysis of the western blot signals revealed significant (p < 0.05, n = 3) differences in LRP expression inWHCO1, MDA-MB 231 and MCF-7 cells, respectively (compared to non-transfected cells). Error bars represent standard deviation.</p

LRP/LR interacts with PS1 of the γ-secretase complex.

<p>(A)(i) FRET analysis with PS1-GFP donor and LRP-dsRed acceptor in HEK293 cells. Distinct FRET signal is detected on the normalized FRET scale. Scale bar: 20 µm. (ii) Enlarged view of single cell from (i) showing that energy is transferred in the cytoplasmic and plasma membrane regions of the cell. (iii) Enlarged view from cell in (i) showing FRET signal occurs in perinuclear membrane. (B) FRET analysis between PS1-GFP donor and LRP-dsRed acceptor performed on IMR-32 cells. Blue FRET signal is detected in the normalised FRET panel indicating an interaction is present between the two proteins. Scale bar: 5 µm. (C) FLAG co-immunoprecipitation assay was performed utilizing lysates of HEK293 cells transfected with LRP::FLAG and PS1-GFP. Immunoblot performed using rabbit monoclonal antibody (EP2000Y) for Presenillin 1. Lane (1) Non transfected cell lysates, (2) cell lysates expressing GFP alone, (3) cell lysates expressing PS1-GFP, (4) FLAG co-immunoprecipitation eluate of assay performed using cell lysates expressing LRP::FLAG and PS1-GFP. Detected bands are 36 kDa indicating PS1 dimers and not PS1-GFP.</p

β-secretase doesn't interact with LRP/LR as revealed by FRET analysis.

<p>(A) FRET did not occur between the donor BACE1-GFP and the acceptor LRP-dsRed in HEK293 cells as no signal was detected on the normalized FRET scale. Scale bar: 20 µm. (B) Similarly, a lack of interaction was detected between LRPdsRed and BACE1-GFP when IMR-32 cells were used. Scale bars:10 µm.</p