Zn(II)PPIX induces apoptosis and increases formation of reactive oxygen species. C-26 cells were grown in Petri dishes for 24 hours before addition of Zn(II)PPIX for the indicated time. [A] Cell cycle analysis was performed by flow cytometry analysis using ethanol-fixed, propidium iodide-stained cells. [B and D] Western blotting for expression of cyclin D1, cleaved caspase 3 or tubulin as a loading control was performed. [C] Induction of apoptosis was evaluated by staining of cells with propidium iodide and annexin V. [E] HO-1 silencing was evaluated with Western blotting. Controls represent expression of HO-1 in hemin-treated C-26 cells. HuHO-1 siRNA is a negative control targeting xenogeneic (human) HO-1 gene, muHO-1 siRNA is a murine sequence that knocks-down HO-1 expression in C-26 cells. [F and G] production of reactive oxygen species was evaluated with flow cytometry by measuring CM-H2DCFDA fluorescence in comparison with controls.