Figure 3
Complement C3 and C5 cleavage fragments enhance migration of leukemic cells through phosphorylation of p38 MAPK-dependent downregulation of heme oxygenase 1 (HO-1). (a) RT-qPCR analysis of human HO-1 transcripts in mRNA samples purified from U937 (left) and KG-1a (right) cell lines cultured with C3a (1 μg/ml), C5a (140 ng/ml) or SDF-1 (300 ng/ml) in serum-free medium for 6 h at 37 °C. β2-microglobulin was used as an endogenous control. Samples containing only water instead of cDNA were used in each run as a negative control. Cells without any stimulation served as a control. *P<0.05 is considered statistically significant between cells exposed to anaphylatoxins or SDF-1 and unstimulated cells. (b) Western blot for human HO-1 in protein lysates collected from U937 (left) and KG-1a (right) cell lines (20 μg per sample). After incubation of cells with C3a, C5a or SDF-1 at the doses indicated above, protein was immediately extracted and later quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) and Multimode Analysis Software (Beckman Coulter Inc, Fullerton CA, USA). In parallel, β-actin was also analyzed to ensure the equivalence of loading. Proteins extracted from cells cultured in only assay medium served as a control. (c) Western blot analysis for phospho-p38 MAPK in protein lysates collected from quiescent U937 (left) and KG-1a (right) cell lines. Cells were stimulated with 0.5% BSA in RPMI 1640 medium, C3a (1 μg/ml), C5a (140 ng/ml) or SDF-1 (300 ng/ml) for 5 min at 37 °C. Total p38 MAPK was also analyzed to ensure equal protein loading in each lane.
