Fig. 5.

Netrin-1 promotes the maintenance and motility of U251MG stem-like cells. a Either wild-type or NTN1 overexpressing U251MG cells were cultured under condition that favor neural stem cell proliferation for 3 weeks. The formed neurospheres were calculated. The number of the neurospheres is presented as relative to the wildtype U251MG. Error bars represent SD. * = p < 0,05. b The formed U251MG neurospheres were collected and the expression of stem cell markers was evaluated with quantitative real time PCR. The mRNA expression is normalized to the expression of GAPDH. The results are expressed as relative to the mRNA expression levels of U251MG cells cultured under standard conditions. Error bars represent the standard deviation. c The expression of the markers was also confirmed with immunofluorescence microscopy. Scale bars represent 50 μm. d U251MG neurospheres were dissociated and plated into Matrigel coated Boyden chambers. Cells were allowed to invade for 8 h and then quantitated. Number of invaded cells are presented as relative to the control group. Error bars represent SEM and * = p < 0,05. e Neurospheres were embedded into 3D Matrigel matrix and monitored for 24 h. The area of the spheroids was measured at the starting and ending point of the experiment. The graphs represent the relative change in the area of the spheroid. Error bars represent SEM and * = p < 0,05. f Images represent the endpoint of the 3D Matrigel invasion assays. The invasion of the control spheroids is presented also on Additional file 4: Video 1 and the invasion of the rNTN1 treated spheroids in Additional file 5: Video2. Scale bars represent 200 μm