Netrin-1 does not affect distal signalling events following C5aR1 ligation.

<p>(<b>A</b>) BMDMs were seeded into 6 well dishes (2x10⁶ cells/dish) and then treated with either vehicle or 3 nM netrin-1 for 45 min at 37°C, 5% CO₂. Cells were then stimulated with either vehicle or 10 nM C5a for 5 or 30 min at 37°C, 5% CO₂. Cell lysates were prepared and western blotting conducted for either phosphorylated Akt (p-Akt) or ERK1/2 (p-ERK1/2), followed by stripping and re-staining for total ERK1/2 as a loading control. Quantification by densitometry of C5a-induced (<b>B</b>) Akt or (<b>C</b>) ERK1/2 phosphorylation. Data are mean + SEM, n = 3 biological replicates. BMDMs were seeded into 96 well black walled plates (5000 cells/well) and then treated with either vehicle or 3 nM netrin-1 for 45 min at 37°C, 5% CO₂. Cells were then stimulated with either vehicle or 10 nM C5a for 5 or 30 min before being fixed and stained with Alexa Fluor® 488 phalloidin to visualise F-actin. Images were then taken using an IncuCyte® ZOOM microscope and the average cell area calculated. Representative images showing phalloidin staining in green and the cell outlines calculated by the IncuCyte® ZOOM software in pink for cells stimulated with (<b>D</b>) vehicle alone, (<b>E</b>) 3 nM netrin-1 alone, (<b>F</b>) 5 min 10 nM C5a alone and (<b>G</b>) 3 nM netrin-1 and 5 min 10 nM C5a. Quantification of average cell area after (<b>H</b>) 5 min or (<b>I</b>) 30 min stimulation. Data are mean + SEM, n = 3–5 biological replicates with 4 technical replicates per condition. (<b>J</b>) BMDMs (2 x 10⁴) were placed into a 96 well E-plate allowed to adhere for 2–3 hours at 37°C, 5% CO₂. Afterwards, cells were treated with either vehicle or 3 nM netrin-1 for 45 min prior to being stimulated with vehicle or 10 nM C5a. (<b>K</b>) Quantification of the change in cell index following stimulation. Data are mean + SEM, n = 4 biological replicates with 3–4 technical replicates per condition. Statistical analysis was conducted by two-way ANOVA with Sidak’s multiple comparisons correction. ns P > 0.05, for indicated comparisons. * P < 0.05, ** P < 0.01, *** P <0.001 compared to the respective vehicle control.