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Files in this Data Supplement:
Fig. S1. Immunofluorescence analysis of endogenous PTEN and PTEN-GFP. Unstimulated and fMLP-stimulated PTEN-GFP-expressing HL60 cells were analyzed by immunofluorescence with anti-PTEN antibodies (red). The green channel corresponds to the fusion protein GFP signal. Four focal planes at the z-axis from the bottom to the top of the cell are shown. The cells are representative of 20/20. Bar, 10 mm.
Fig. S2. Analysis of expression levels of PTEN chimeras. (A) Equal amounts of cell lysates from untransfected HL60 cells, HL60 cells transfected with PTEN-GFP (used in Fig. S1), GFP-PTEN (used in Fig. 3) and PTEN-C/S-GFP (used in Fig. 4) were resolved in SDS-PAGE, and PTEN and tubulin (loading control) were analyzed by western blot. After densitometry, the ratio between ectopically and endogenously expressed PTEN was calculated, then corrected for transfection efficiency as estimated by FACS analysis. (B) Western blot analysis of PTEN and tubulin in lysates from Jurkat cells transfected with the indicated PTEN chimeras and untransfected HL60 cells.
Fig. S3. PTEN-GFP chimera dynamics during Jurkat cell chemotaxis. Chemotaxis of Jurkat cells co-expressing PTEN-GFP and RFP towards a CXCL12 gradient was analyzed. Dark-phase as well as red and green fluorescence were recorded in a confocal microscope; frames were recorded every 20 sec. Dark-phase, green and red fluorescence are shown for representative frames. The cell is representative of 14/15. Bar, 10 mm.
Fig. S4. PTEN-C/S-GFP chimera dynamics during Jurkat cell chemotaxis. Jurkat cells co-expressing PTEN-C/S-GFP and RFP were analyzed in chemotaxis assays towards CXCL12. Dark-phase and red and green fluorescence were recorded in a confocal microscope; frames were recorded every 20 seconds. Representative frames are shown. The cell is representative of 12/12. Bar, 10 mm.
Fig. S5. PI3K inhibitors impair Jurkat cell chemotaxis. Serum-starved Jurkat cells were pretreated (1 hour, 37°C) with LY294002 at indicated doses or with DMSO (untreated), then tested for chemotaxis towards CXCL12 in transwell assays. After 90 minutes, cells in the lower chamber were counted in a flow cytometer. Results are expressed as the percentage of migrated cells.
Movie 1. PTEN-GFP chimera dynamics during HL60 cell chemotaxis. fMLP-induced chemotaxis of DMSO-differentiated HL60 cells expressing PTEN-GFP (green) and the inert cytosolic RFP (red). The movie shows the homogeneous distribution of both markers. The first 20 frames were recorded every 5 seconds, then every 20 seconds (display rate: 2 frames/second).
Movie 2. PTEN-GFP chimera and RFP-GPI dynamics during HL60 cell chemotaxis. fMLP-induced chemotaxis of DMSO-differentiated HL60 cells expressing PTEN-GFP (green) and the polarization marker RFP-GPI (red), a raft-associated protein. The movie shows that PTEN-GFP is evenly distributed in the cytosol, whereas RFP-GPI accumulates at the leading edge and the uropod of moving cells. The first 20 frames were recorded every 5 seconds, then every 20 seconds (display rate: 2 frames/second).
Movie 3. GFP-PTEN chimera dynamics during HL60 cell chemotaxis. fMLP-induced chemotaxis of DMSO-differentiated HL60 cells expressing GFP-PTEN (green) and the inert cytosolic RFP (red). The movie shows the homogeneous distribution of both markers. The first 20 frames were recorded every 5 second, then every 20 seconds (display rate: 2 frames/second).
Movie 4. PTEN-C/S-GFP chimera dynamics during Jurkat cell chemotaxis. fMLP-induced chemotaxis of DMSO-differentiated HL60 cells expressing PTEN-C/S-GFP (green) and the raft marker RFP-GPI (red). The movie shows that PTEN-C/S-GFP is evenly distributed in the cytosol, whereas RFP-GPI is accumulated at the leading edge and the uropod of moving cells. The first 20 frames were recorded every 5 seconds, then every 20 seconds (display rate: 2 frames/second).
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