Fig. 3

SDF-1 and CCL21 chemokines activate mTORC1 pathway, promote tumor proliferation and up-regulate the Ki67 expression in H9 cell line and primary SS cells. a left panel, serum-starved H9 cells were left untreated (UNT) or pre-incubated with rapamycin for 2 h and then stimulated or not with SDF-1 or CCL21 for 30 min at indicated concentrations. WB was performed for the specified proteins. a Right panel, densitometric data normalized to β-actin were expressed as mean ± SEM of three independent experiments. Graph shows phospho protein levels expressed as FC respect to UNT samples *P ≤ 0.02, **P = 0.05; °°P ≤ 0.0001; b upper panel, SS cells from patients cultured in complete medium at high density, left UNT or pre-incubated with 30 nM of rapamycin for 2 h and then stimulated or not for 30 min with 100 ng/ml of SDF-1 or b lower panel,with 10 ng/ml of CCL21. WB was performed for the specified proteins. Because of the timing of patient recruitment, the gels were run at separate times and the lanes were composed for the figure. c upper panel, SS cells were cultured in vitro at high density for 5 days in complete medium used alone (NT) or supplemented with 300 ng/ml of SDF-1 or CCL21 in presence or absence of rapamycin used at 30 nM or IL2 at 30U/ml plus IL7 at 10 ng/ml. Cell proliferation was determined by MTT assay and data are expressed as means of OD values ± SEM. Statistics were calculated as paired t-test. *P = 0.05; **P = 0.01; c lower panel, cell-activation induced by 300 ng/ml of SDF-1 was evaluated in SS cells obtained from SS94 patient, by Ki67 detection using FACS within neoplastic clone recognized by CD3,CD4, CCR7 and TCR-Vβ1 chain rearrangement