Fig 2: Effects of 4-HPR treatment on AF1q expression in a panel of cancer cell lines.Western blot analysis for AF1q expression in OVCA432 and SKOV-3 (A) cells, in T47D and SK-N-BE (B) cells, and in OVCAR-3 and HeLa (C) cells treated for 24 hours with 4-HPR at the indicated doses. As a control for loading, the blots were incubated with actin antibody.

Fig 3: Relationship between AF1q upregulation and 4-HPR-induced signaling cascade.Western blot analysis for AF1q expression in A2780 cells treated for 24 hours with 5 µM 4-HPR, with or without 100 µM vitamin C (A), 10 µM salubrinal (B), or 10 µM SP600125 (C). (D) Western blot analysis for AF1q expression in A2780 cells stably transfected with a plasmid containing a PLAB siRNA or a scrambled nonsilencing siRNA following addition of 5 µM 4-HPR for 24 hours. As a control for loading, the blots were incubated with actin antibody.

Fig 4: Effect of AF1q overexpression in the onset of basal apoptosis.(A) Immunofluorescence analysis of A2780 cells transiently transfected with GFP alone (upper panels) or GFP-tagged AF1q vector (AF1q-GFP) (lower panels). After 48 hours, GFP and AF1q-GFP cells were stained with Hoechst 33342 and nuclear morphology was examined with a fluorescent microscope. Cells of interest are marked by arrows. Cells with condensed and fragmented nuclei were identified and scored as apoptotic cells. One experiment representative of three is shown. The scale bar represents 10 µm. (B) Apoptosis was represented as percentage of apoptotic cells per 100 green fluorescent cells (at least 200 cells per sample) in A2780 (left panel) and OVCAR-3 (right panel) cells transfected as in (A). Data represent the mean±S.D. of three independent experiments. Asterisks indicate significant difference (P<0.05).

Fig 5: Effects of 4-HPR on AF1q expression in A2780 and A2780/HPR cells.Western blot analysis for AF1q expression in A2780 (A) and A2780/HPR (B) cells treated for 24 hours with 5 and 10 µM 4-HPR. As a control for loading, the blots were incubated with actin antibody.