Fig. 4.
Effects of HMGB1 secreted from4T1 BC cells on neurite outgrowth of DRG SN cells. (A) Effects of co-culture with 4T1 BC cells on neurite outgrowth of F11 SN cells in microfluidic culture platforms. F11 SN cells (105 cells, left chambers) were co-cultured with 4T1 BC cells (106 cells, right chambers) for 48 h in the absence or presence of the HMGB1 neutralizing antibody (50 μg/ml) or control IgY antibody. The chambers were then labeled with calcein AM (1 μM) for 10 min and F11 SN cells with extended neurites were observed under a fluorescent microscope. The number of F11 SN cells exhibiting extended neurite was increased in the co-cultures with 4T1 BC cells. Scale bar 150 µm. (B) Quantitative evaluation of neurite outgrowth in F11 SN cells in microfluidic culture platforms seen in Fig. 4A. F11 SN cells with extended neurite longer than 50 μm were counted under a fluorescent microscope using Neuron J software and are shown on Y-axis in the figure. The number of F11 SN cells with extended neurite is significantly increased in the co-cultures with 4T1 BC cells, and HMGB1 antibody significantly decreased the number. Data are shown as mean ± SD (n = 4). * Significantly different from medium control (p < 0.05). # Significantly different from 4T1 or 4T1 + IgY (p < 0.05). (C) Effects of CM harvested from 4T1 BC cell cultures on neurite outgrowth of F11 SN cells. F11 SN cells (104 cells/24-well plates) were treated with 4T1 BC cell CM (20%, v/v) in the neuron growth medium in the absence or presence of the neutralizing antibody to HMGB1 or control IgY antibody for 5 days. The plates were then stained with calcein AM (1 μM) for 10 min and observed under a fluorescent microscope. DbcAMP (1 mM) is a positive control. Scale bar 20 µm. (D) Quantitative evaluation of the effects of CM harvested from 4T1 BC cell cultures on neurite outgrowth of F11 SN cells. The length of all fluorescent neurites outgrowing from the cell body of F11 SN cells, and the number of all fluorescent cell body of F11 SN cells in plates were determined using Neuron J software under a fluorescent microscope. Neurite length (μm/cell) on Y-axis of the figure was calculated as, total length of all outgrowing neurites/number of total cell body of F11 SN cells. Neurite length of F11 SN cell expressed on Y-axis as μm per cell was increased to 123.73 ± 6.67 μm (Mean ± SD) by treatment with 4T1 CM, which is decreased to 37.61 ± 13.78 μm (Mean ± SD) by HMGB1 antibody. Data are shown as mean ± SD (n = 4). * Significantly different from medium control (p < 0.05). # Significantly different from 4T1 and 4T1 + IgY control antibody (p < 0.05).