FIGURE 1.

Pro-inflammatory cytokines induce transcription of the C3 and CFB genes and secretion of the C3 and FB proteins in ARPE-19 cells. ARPE-19 were treated with 10 ng/ml TNF-α, 10 ng/ml IL-1β, or vehicle alone for 24 h, and RNA levels of (A) C3, (B) CFB, (D) complement factor H (CFH), (E) CD46, (F) CD55, and (G) CD59 were measured by qRT-PCR. Data were normalized based on the mRNA levels from non-stimulated cells (ns; 2^{−ΔCT gene − GAPDH} values were in the range 0.00–0.04 for non-stimulated cells), and expressed as mean ± SEM (n = 5–10 from as many independent experiments). Statistical analysis was carried out using the Kruskall-Wallis test followed by Dunn’s multiple comparison test. (C) Presence of the C3 and FB proteins in cell culture media was assessed by western blotting. Proteins were separated by SDS-PAGE in reducing conditions, transferred onto PVDF membranes, and C3 (i.e., both α and β chains) and FB were revealed by appropriate polyclonal antibodies. Gels are shown that are representative of two independent experiments. Positions and apparent molecular weights of the C3α, C3β and FB bands are indicated.