Fig. 5

Growth, migration and ERK-activation after complement activation. (a) Growth of sub-confluent HN5 cells was measured at 3 and 24 h after adding NHS or HIS, using an SRB assay. Growth of control cells treated with medium only is set to 100%. No significant difference is found between HIS and NHS treatment, indicating complement activation did not affect growth. (b) A scratch was made in a monolayer of HN5 cells, and migration of cells into the open area was measured 24 h after adding NHS, HIS or medium only. The area of the original scratch at 0 h is set to 100%. (c) Using qPCR, induction of growth and immune related genes was tested after 48 h of Iressa treatment and a subsequent 3 h complement activation using NHS. In cell lines HN5 and HN4, no significant difference in induction is seen when compared to HIS set to 1. (d) HNSCC cell lines were treated with Iressa for 48 h and subsequently incubated with 10% NHS or HIS, cell lysates were then blotted for phosphorylated ERK (P-ERK) and normalized to total ERK (T-ERK). Bars represent the ratio of ERK phosphorylation after incubation with NHS compared to HIS, average of 3 experiments is used. (e) ERK phosphorylation was further tested in HN5 cells after incubation with C1q depleted serum, and C1q depleted serum reconstituted with C1q (100 μg/mL),and was shown with a representative blot. Error bars represent SEM, * p < 0.05