Fig. 2

EGF and NTN4 delayed cell senescence induced by H₂O₂ and TMZ. (a) The expression levels of NTN4 were determined by Q-RT-PCR in U251MG cells overexpressing NTN4 (NTN4.pcDNA3) and in mock control U251MG cells (Mock). (b) Five days after H₂O₂ treatment, mock cells and NTN4.pcDNA3 cells were fixed and analyzed by beta-gal staining. H₂O₂ triggered senescence of U251MG cells, while NTN4 overexpression significantly decreased the number of senescent cells induced by H₂O₂. (c) After the H₂O₂ treatment, U251MG cells were treated with EGF (40 ng/mL) every two days. On the 4th and 7th day after EGF treatment, U251MG cells were fixed and analyzed by the beta-gal staining. Four days after EGF treatment, the number of senescent U251MG cells was significantly less compared with control cells. Seven days after EGF treatment, the number of senescent U251MG cells was ~ 2-fold less than in EGF non-treated groups. (d-f) After TMZ treatment, U251MG cells were treated with EGF (40 ng/mL), NTN4 (50 ng/ml), and EGF + NTN4, separately. All the proteins were re-supplied every two days. After treatment for 4 days, TMZ strongly induced senescence in U251MG cells (d), GBM-14042 (e) and GBM-112D cells (f), while EGF, NTN4, and EGF + NTN4 attenuated the pro-senescence effect of TMZ. Mean ± SE, n ≥ 3, * p-value < 0.05, ** p-value < 0.01