Figure 9

Knockdown of GM-CSF protein levels after siRNA application in cancer cells. HeLa/DLD-1 cells were transfected with control siRNA (1/1*, 2/2*) or GM-CSF siRNA (3/3*, 4/4*) and cultured in the absence or presence of 25 ng/mL HB-EGF. The numbers indicate the culture conditions and the corresponding supernatants (SN) used for ELISA or cell stimulation. (A) Blockade of GM-CSF production in cultures of HeLa/DLD-1 cells transfected with GM-CSF siRNA was confirmed by immunocytochemistry (2/2* vs. 4/4*) and ELISA (left side; 2/2* vs. 4/4*, p < 0.05). (B) SN from GM-CSF-silenced HeLa/DLD-1 did not induce HB-EGF expression in mononuclear phagocytes (Mø), as revealed by flow cytometry (2/2* vs. 4/4*) and ELISA (left side; 2/2* vs. 4/4*, p < 0.05). (C) Mø stimulated with SN from GM-CSF-silenced HeLa/DLD-1 cells released SN less effective at inducing GM-CSF in non-silenced cancer cells, as determined by ELISA (see Methods section; SN2 vs. SN4, p < 0.05). Representative pictures or the means ± SD out of 5 experiments are shown.