Fig 2: The effect of exosomal S1P2 on ERK-1/2 signaling and S1P2 processing in MEFsMEFs quiescent for 24 hr were treated for 15 min with exosomes isolated from CM of MDA-MB-231 cells that had been treated with either scrambled siRNA or S1P2 siRNA for 24 h. (A) Western blot showing the effect of exosomes (EXO, μl) from scrambled siRNA (100 nM) and S1P2 siRNA (200 nM)-treated MDA-MB-231 cells on ERK-1/2 activation in MEFs. Blots were probed with anti-phospho ERK-1/2 antibody. (B) Western blot showing the uptake into MEFs of the short S1P2 (Mr = 36 kDa) form from exosomes (EXO, 50 or 100 μl) isolated from MDA-MB-231 cells. Blots were probed with anti-S1P2 antibody. (A, B) Blots were re-probed with anti-actin antibody to ensure equal protein loading. Results are representative of 2-3 independent experiments.

Fig 3: Uptake of exosomal S1P2 from breast cancer cells and processing by MEFsMEFs quiescent for 24 hr were treated with NCM or CM from MDA-MB-231 cells for 10 min. MEFs were pre-treated with and without JTE-013 (10 μM) or CYM5520 (10, 25 μM) for 15 min prior to addition of NCM or CM. (A) Western blot showing the lack of effect of CYM5520 alone on ERK-1/2 activation in MEFs. (B) Western blot showing the uptake into MEFs of the short S1P2 (Mr = 36 kDa) form and the activation of ERK-1/2 in response to CM isolated from MDA-MB-231 cells. (A) and (B) Blots were re-probed with anti-actin antibody to ensure equal protein loading. (C) Immunofluorescence images of control, CM-stimulated and CYM5520 (10 μM)-stimulated MEFs co-stained with anti-actin (TRITC secondary antibody, red) and anti-S1P2 (FITC secondary antibody, green) antibodies. (D) Fluorescence image of MEFs showing uptake of GFP-hsp70 by detection of GFP and co-stained with anti-actin antibody using a Texas Red conjugated secondary antibody or DAPI. Results are representative of 3 independent experiments.

Fig 4: Identification of S1P2 in exosomes shed from MDA-MB-231 breast cancer cells(A) Western blot with anti-GFP or anti-mCherry antibodies showing the over-expression of GFP-hsp70 and mCherry-tgs101 in vector (V) or plasmid transfected MDA-MB-231 cells for 24 or 48 h. (B) Western blot with anti-GFP or anti-mCherry or anti-HA antibodies showing the presence of GFP-hsp70, mCherry-tsg101 or HA tagged S1P2 (Mr = 40 kDa) in transfected MDA-MB-231 cell lysate (CL) and acid precipitates (PPT) of CM. (C) Western blot with anti-hsp70, anti-CD63 and anti-S1P2 antibodies showing the presence of GFP-hsp70, CD63 or S1P2 in MDA-MB-231 cell lysates (CL) and isolated exosomes (EXO) from CM. (D) Immunofluorescence image of MDA-MB-231 cells stained with anti-CD63/TRITC (red) secondary and anti-S1P2/FITC (green) secondary antibodies showing co-localisation (yellow) of CD63 and S1P2 in large vesicles typical of MVBs. (E) Electron micrograph of immunogold staining with anti-CD63 [attached to secondary goat anti-mouse IgG-immune-gold particles (15 nm)] and anti-S1P2 antibodies [attached to secondary goat anti-rabbit IgG-immune-gold particles (10 nm)] in exosomes isolated from CM of MDA-MB-231 cells. Control represents exosomes that have not been incubated with primary antibody. Results are representative of 3 independent experiments.

Fig 5: Identification of S1P4 and S1P2 shed from breast cancer cells(A) Western blot with anti-S1P4 antibody showing the presence of S1P4 in MDA-MB-453 cell lysate (453CL) and acid precipitates (PPT) of conditioned medium (CM) isolated after 48 h incubation with opti-MEM. (B) Western blot with anti-S1P2 antibody showing the presence of S1P2 in MDA-MB-231 cell lysate (231CL) and acid precipitates (PPT) of conditioned medium (CM) isolated after 48 h incubation with opti-MEM. (C) Western blot with anti-S1P4 antibody showing the absence of S1P4 in acid precipitates (PPT) of conditioned medium (CM) isolated from MDA-MB-231 cells after 48 h incubation with opti-MEM. The presence of S1P4 in cell lysates (231CL) of MDA-MB-231 cells is also shown. (D) Western blot with anti-S1P2 antibody showing the absence of S1P2 in acid precipitates (PPT) of conditioned medium (CM) isolated from MDA-MB-453 cells after 48 h incubation with opti-MEM. The presence of S1P2 in cell lysates (453CL) of MDA-MB-453 cells is also shown. Results are representative of 3 independent experiments.