Fig 2: SEMA3F suppresses VEGF through the inhibition of mTOR-Akt signals. A,B, U87MG cells were transiently co-transfected with a full-length human VEGF promoter luciferase plasmid and a pGL4.74[hRluc/TK] plasmid as an internal control. Cells were treated with SEMA3F (640 ng/ml for 30 minutes) prior to the addition of DFO (250 µM) or the culture of cells in a hypoxia chamber (1% O2). After 18 hours, VEGF promoter luciferase activity was analyzed. C, U87MG cells were transiently co-transfected with our VEGF promoter luciferase and pGL4.74[hRluc/TK] plasmids and with either a pcDNA3.1 empty vector or our constitutively active Akt (2DAkt). The cells were treated with SEMA3F for 30 minutes prior to the addition of DFO. After 18 hours, VEGF promoter luciferase activity was analyzed. D, Parental U87MG cells were treated with SEMA3F, rapamycin (10 nM), Torin 1 (10 nM) alone or in combination as indicated for 30 minutes prior to the addition of DFO, and culture supernatants were collected after 18 hours; VEGF protein levels were analyzed by ELISA. In each panel data are representative of 3 independent experiments. Bar graphs represent the mean ± SD of n = 3 experiments performed in triplicate, *p < 0.01 vs. control.

Fig 3: SEMA3F inhibits the phosphorylation of Akt, mTOR and S6K.A, U87MG cells untreated (control) or following treatment with SEMA3F (640 ng/ml) for 30 minutes. Cell lysates were evaluated by phosphoprotein kinase antibody array. The intensity of each dot/phosphoprotein was measured using Image J software, as shown in Supplementary Table 1. B, Results of the array were validated by Western blot analysis. C, U87MG cells were treated with SEMA3F (640 ng/ml) as a time course up to 60 minutes and were analyzed by Western blot. D, U87MG cells were transfected with a control siRNA or with a NRP2-specific siRNA (20 nM). After 48 hours, the cells were treated with SEMA3F (640 ng/ml) for 30 and 60 minutes and analyzed by Western blot. Panels B–D are representative of 3 independent experiments. E, U87MG, Jurkat and HUVEC cells were treated with SEMA3F (200, 600, 1800 ng/ml, bars from left to right) for 15 minutes (grey bars) or 30 minutes (black bars); as a positive control, HUVEC were treated with VEGF-A (25 ng/ml) for 15 and 30 minutes. In addition, HUVEC were pre-treated with SEMA3F (1800 ng/ml) or PBS as a control for 30 minutes and subsequently VEGF-A (25 ng/ml) was added to the culture for 15 and 30 minutes. PI-3K activity was analyzed by ELISA according to the manufacturer’s instructions. Data represent the mean ± SD of 3 experiments.

Fig 4: SEMA3F disrupts both mTORC1 and mTORC2 complex formation.A, U87MG cells were treated with SEMA3F (640 ng/ml) for 30 minutes and were subjected to immunoprecipitation and Western blot analysis with anti-mTOR, -raptor and -rictor as illustrated. B, U87MG cells were treated with rapamycin (10 nM) or Torin 1 (10 nM) for 30 minutes, prior to SEMA3F (640 ng/ml) treatment for 60 minutes; lysates were analyzed by Western blot. C, The right bar graphs represent densitometric analysis of the illustrated blot showing the fold change in intensity (mean ± SD) relative to the untreated control (*p < 0.01; **p < 0.001 vs. untreated control). D, U87MG cells were transiently transfected with a pcDNA3.1 empty vector or with constitutively active Akt (2DAkt). Cells were treated with SEMA3F (640 ng/ml) and lysates were analyzed by Western blot. All data are representative of 3 independent experiments.

Fig 5: mTORC2 participates in SEMA3F-induced RhoA inactivation and loss of stress fibers. A, U87MG cells were treated with SEMA3F (640 ng/ml), rapamycin (10 nM) or Torin 1 (10 nM) for 30 minutes. Subsequently, cells were stained with Alexa Fluor 488 phalloidin and Hoechst 33342 to identify F-actin cytoskeleton stress fibers and cellular nuclei, respectively. Representative cellular staining of is shown in each panel; the bar graph shows the mean ± SD number of fibers/cell in an average of 3 independent experiments. The scale bar indicates 20 µm. B, U87MG cells were transiently transfected with a pcDNA3.1 empty vector or with a wild type (WT) mTOR plasmid and after 18 hours were treated with SEMA3F (640 ng/ml) for 30 minutes. Cells were stained as described above in A. Representative cellular staining is shown; bar graph represents the number of fibers/cell (mean ± SD) from 3 independent experiments. C, U87MG cells were transfected with control siRNA or with raptor- or rictor-specific siRNAs (20 nM). After 48 hours, they were treated with SEMA3F for 30 minutes and stained with Alexa Fluor 488 phalloidin and Hoechst 33342 as above. The number of stress fibers was evaluated in 3 independent experiments and shown as the mean ± SD. D, U87MG cells were transiently transfected with pcDNA3.1 empty vector or with our WT mTOR plasmid. After 18 hours, the cells were treated with SEMA3F (640 ng/ml) for 10 minutes and RhoA activity was evaluated. E, U87MG cells were transfected with control siRNA or with raptor- or rictor-specific siRNAs (20 nM), were treated with SEMA3F (640 ng/ml) for 10 minutes and RhoA activity was analyzed. In Panels D–E, the intensity of active RhoA was normalized to respective total RhoA; the numbers below each gel lane represent the fold-change in intensity relative to control. Panels D–E are representative of 3 independent experiments.