Fig 2: NTSR2 phosphorylation in B-CLL and recruitment of Gia proteins. (a) Representative western blot analysis of Src phosphorylation in MEC-1 cells overexpressing NTSR2 (pCMV6-NTSR2) in the presence or absence of BDNF (100 ng/ml), pertussis toxin (PTX, 200 ng/ml) or K252a (100 nM) for 24 h. (b) Ratio of phosphorylated Src vs pan-Src protein, normalized against actin. Values are means±s.e.m. of three independent experiments in a.u. (c, d). After immunoprecipitation (IP) of NTSR2 from MEC-1 cells overexpressing NTSR2 (pCMV6-NTSR2) or not (EV), or from B-CLL cells in the presence or absence of BDNF (100 ng/ml) or NTS (40 µM/ml), the immunocomplexes were immunoblotted (IB) with anti-Gia1/2 antibodies. (e, f) After immunoprecipitation (IP) of anti-pan-phosphoprotein was performed on MEC-1 cells overexpressing NTSR2 and B-CLL cells in the presence or absence of BDNF (100 ng/ml) or SR142948A (67 µM), the phosphorylation of NTSR2 was detected by immunoblotting (IB) with anti-NTSR2 antibodies. (g) Apoptotic ratio, assessed by cell death ELISA, in B-CLL in the presence or absence of SR142948A (67 µM) for 24 h. Values are mean ratios of apoptotic cells (±s.e.m.) of three independent experiments from different patients (n=3). Significant P-values are indicated in the graphs *P<0.05, ***P<0.001.

Fig 3: NTSR2 overexpression induces cell survival signaling pathways. (a,b) Western blot analysis of NTSR2, Src, SAPK/JNK, p38MAPK, and Akt expression from lysates of BL-41 and MEC-1 cells overexpressing NTSR2 (pCMV6-NTSR2) or empty vector (EV) cultured for 24 h post-transfection in basal conditions (10% fetal bovine serum). (c) Apoptotic ratio following NTSR2 overexpression for 24 h, assessed by cell death ELISA, expressed as fold change vs empty vector (EV). (d, e) Representative western blot analysis of Bcl-xL and Bcl-2 expression in BL-41 and MEC-1 cells overexpressing NTSR2 for 24 h. (h, i) Fold change in Bcl-xL or Bcl-2 expression in BL-41 and MEC-1 cells overexpressing NTSR2 for 24 h, normalized against actin, in comparison with EV. (f–i) Similar analyses performed in cells deprived of serum for 24 h. Significant P-values are indicated in the graphs *P<0.05, **P<0.01, ***P<0.001.

Fig 4: NTSR2 interacts with the tyrosine kinase receptor TrkB. (a) Quantitative analysis of the level of NTRK2 mRNA (encoding TrkB) in normal B (n=15) and B-CLL (n=30) lymphocytes. Data are expressed as mean fold change in NTRK2 expression (±s.e.m.) vs normal B cells. (b) Representative analysis of TrkB expression from B-lymphocyte cell lysates from two normal donors (D1, D2) or two B-CLL patients (P1, P2). (c) TrkB expression level, normalized against actin, in normal B (n=6) and B-CLL (n=6) cells. Values are means±s.e.m. of three independent experiments. (d) Confocal microscopy analysis of NTSR2 (green) and TrkB (red) and their colocalization in B-CLL cells (yellow staining in merged image, insets 1-1 to 2-2) in the presence or absence of BDNF (100 ng/ml). (e) Mander’s overlap coefficient indicating colocalization of NTSR2 and TrkB after BDNF (100 ng/ml) treatment (means±s.e.m. of three independent experiments). (f) After immunoprecipitation (IP) of TrkB and NTSR2 from B-CLL protein lysates, immunocomplexes were immunoblotted (IB) with the indicated antibodies. (g) Representative cytogram showing co-detection of NTSR2 and TrkB by flow cytometry in B-CLL lymphocytes. Significant P-values are indicated in the graphs **P<0.01, ***P<0.001.

Fig 5: Protective role of BDNF against B-CLL apoptosis. (a) BDNF concentration, quantified by ELISA, in B-CLL patient plasma (n=17, gray boxes) in comparison with healthy donor plasma (n=9, white boxes). (b) Quantitative analysis of BDNF mRNA level in normal B (n=15) and B-CLL (n=30) cells. Data are expressed as mean fold change in expression (±s.e.m.) vs normal B cells. (c) Representative western blot analysis of pro-BDNF and mature BDNF (mBDNF) expression in normal B and B-CLL supernatants after 24 h of culture post-isolation. (d) Western blot analysis of P-Src, Bcl-xL and Bcl-2 in B-CLL cell lysates from two patients after addition of BDNF (100 ng/ml) for 24 h. (e, f) Expression levels of p-Src (e), Bcl-xL and Bcl-2 (f) represented as, respectively, the ratio of phosphorylated Src vs pan-Src protein and the ratios of Bcl-xL and Bcl-2 to actin (B-CLL, n=3). Values are means±s.e.m. of three independent experiments, in a.u. (g, h) Apoptotic ratio, assessed by cell death ELISA, in B-CLL cells cultured with or without 100 ng/ml BDNF for 24 h (B-CLL, n=4) in the presence or absence of siRNA against NTSR2 or control siRNA. Values are the mean ratio of apoptotic cells (±s.e.m.) from four independent experiments. Significant P-values are indicated in the graphs *P<0.05, **P<0.01, ***P<0.001.