Fig 2: PTEN-GFP redistributed to the posterior plasma membrane of electrotaxing cells, which is depended on actin polymerization. a, b Dictyostelium cells did not show the membrane recruitment of PTEN-GFP in the absence of EF. (c–e) An applied EF induced asymmetrical redistribution of PTEN-GFP to the posterior of Dictyostelium cells. (f and g) Latrunculin A treatment abolished asymmetrical redistribution of PTEN, while plasma membrane recruitment was still maintained (see J, ##: P < 0.01, compared between Latraculin A treated and control. h and i Washout latrunculin A restored the asymmetrical redistribution of PTEN. b, e, g and i representative line scan of fluorescence intensity of PTEN-GFP for marked cells in a, d, f and h, respectively. (j) GFP intensities at the plasma membrane were determined relative to that in the cytosol. Values represent the mean ± s.d. **, P < 0.01 compared between the posterior membrane of no EF vs EF treated group; $$, P < 0.01 compared between posterior and anterior membrane within EF treated cells; ##, P < 0.01 compared between anterior within “LatA + EF” and “EF only” group; ns, P > 0.05 compared between the posterior and anterior membrane of “no EF” and “LatA + EF” group. k Membrane fraction and the whole-cell lysates from cells treated with the indicated duration of EFs were analyzed by immunoblotting with antibodies against phospho-PTEN (pS380) or GFP (PTEN-GFP), respectively. l The PTEN proteins were immunopurified from Dictyostelium cells treated with varies duration of EF, and phosphatase activities were measured. *P < 0.05; **P < 0.01 compared with no EF group. n ? 3

Fig 3: PTENL320S and PTENT277A show decreased activities to suppress PIP3 signalling. (a) HEK293T cells expressing GFP fused to the indicated PTEN molecules were examined using fluorescence microscopy. (b) Quantification of the membrane-associated PIP3 level using the PIP3 biosensor RFP-PHAKT. Fluorescence intensity was measured as the ratio between membrane intensity and cytosol intensity.37 Values represent mean±s.e.m. (n=20). (c) Correlations between total PTEN-GFP level and the membrane-bound RFP-PHAKT fraction in HEK293T cells expressing PTENWT-GFP, PTENL320S-GFP, PTENdelEx3-GFP, and PTENT277A-GFP (n=15 for each PTEN-GFP protein).

Fig 4: Chemerin activity involves the PTEN-AKT-FOXO1 axis. a Representative images of retinas of P6 control and K5-Chem mice, injected intraperitoneally with DAPT or vehicle, after staining with IsoB4. Scale bars: 200 µm. b–d Quantification of the vessels area, total vessels length, and junctions density in the remodeling plexus of these retinas (n = 6 for controls vehicle, K5-Chem vehicle and K5-Chem DAPT, n = 5 for controls DAPT). e–g Quantification of the vessels area, total vessels length, and junctions density in the remodeling plexus of retinas from control and K5-Chem mice injected with bpV(HOpic) or vehicle (n = 7 for controls vehicle, n = 6 for K5-Chem vehicle, n = 4 for controls bpV, n = 5 for K5-Chem bpV). h–j Quantification of the vessels area, total vessels length, and junctions density in the remodeling plexus of retinas from control and K5-Chem mice injected with AS1842856 (AS) or vehicle (n = 4 for controls vehicle and K5-Chem AS, n = 6 for K5-Chem vehicle and controls AS). k Representative image of HUVECs stained for CMKLR1. Scale bar: 20 µm. l Representative images of HUVECs treated or not with CoCl2 and stained by DAPI and for HIF1a. Scale bars: 20 µm. m Representative Western blot for HIF1a detection in HUVECs treated or not with CoCl2 and/or 10 nM chemerin. n Quantification of HIF1a normalized to actin. o Representative Western blots of HUVECs treated or not with CoCl2 and/or 10 nM chemerin. p–r quantification of the phosphorylation ratios of PTEN, AKT, and FOXO1. P values versus controls by 2-tailed unpaired Student’s t-test. All data are shown as mean ± SEM. Each point represents 1 animal

Fig 5: L320S and T277A open the conformation of PTEN. (a) PTEN-GFP proteins purified from HEK293T cells were incubated with trypsin for 10 min and analysed by immunoblotting with anti-PTEN antibodies. (b) Quantification of the band intensity of full-length PTEN-GFP. Values represent mean±s.e.m. (n=4). (c) The indicated GFP-PTEN proteins were expressed in HEK293T cells along with HA-ubiquitin and were immunoprecipitated using beads coupled to anti-GFP antibodies. The immunoprecipitated proteins were analysed by immunoblotting with antibodies to GFP and HA. (d) Phosphorylation of PTEN was quantitatively analysed by immunoblotting whole cell lysates carrying various PTEN constructs. Values represent mean±s.e.m. (n=3). (e) Whole-cell lysates from HEK293T cells expressing the indicated GFP-PTEN proteins were incubated with the C-terminal tail to create PTEN352–403-YFP-FLAG, which was immunoprecipitated using beads coupled to anti-FLAG antibodies and analysed by immunoblotting.