Fig. 6.
EDP-induced blebbing involves the RPSA elastin-laminin receptor. Identification of the RPSA protein as the EDP receptor by affinity chromatography. a Eluted samples were submitted to silver staining and b western blot using RPSA antibodies. c Optical section realised by confocal microscopy of HT-1080 cells incubated with Tamara-AG9 (AG9) at 5 × 10−5 M for 1 h at +4 °C and after immunolocalisation of RPSA protein. Colocalisations were studied with Colocalisation plugin of ImageJ. Scale bar: 20 µm. d Blebbing quantification in HT-1080 cells in the presence or not of RPSA-blocking monoclonal antibody (10 µg/mL) evaluated by counting 10 fields per well under a phase contrast optical microscope after EDPs and AGVPGLGVG stimulations for 40 min. Data from one experiment, representative of three independent experiments, are shown. Results (mean ± S.E.M) were expressed as percentage of control (EDPs untreated cells). **p < 0.01, ***p < 0.001. e Real-time PCR analysis of RPSA 48 h after treatment with RPSA siRNA vs negative control siRNA-treated cells (Ctl siRNA). Results (mean ± S.E.M; n = 3) were expressed as the ratio of RPSA mRNA to EEF1a1 mRNA. f RPSA siRNA transfected HT-1080 cells incubated with TAMRA-AGVPGLGVG (AG9) at 5 × 10−5 M for 1 h at +4 °C. Confocal imaging was realised to study the TAMRA-AGVPGLVGV and RPSA localisations on HT-1080 cells. Scale bar: 10 µm. g RSPA expression was analysed in different cell types by western blot. Data from one experiment, representative of three independent experiments, are shown. h Quantitative evaluation of RPSA expression by different cell types. i Correlation between Δblebbing and ratio RPSA/actin in different cell types. Δblebbing = EDP-dependent blebbing – unstimulated blebbing. Linear regression test was performed for each analysis and the R and p values are indicated in the graph. j Optical section obtained by confocal microscopy of control HT-1080 cell and EDP-induced blebbing HT-1080 cell to analysis RPSA localisation; western blot for RPSA realised with 10 µg of HT-1080 cell extract and extracellular vesicle proteins. Scale bar: 10 µm