Figure 4

(A) Growth properties of IGFBP3 treated HB cells. The viability of tumor cells in the presence (IGFBP3) or absence (control) of 1 μg/ml recombinant human IGFBP3 was assessed at the time points indicated using MTT assays and optical density (OD) measurements. The values given represent the mean ratio of treated versus untreated cells from triplicate measurements ± SEM. (B) Annexin V staining. IGFBP3-treated cells were analyzed for phosphatidylserine membrane asymmetry using Cy5-conjugated annexin V and calcein staining. Representative plots of the flow-cytometric measurements depicting the percentages of apoptotic cells (annexin V and calcein positive) in the control (left panel) and recombinant human IGFBP3 treated cells (right panel) are shown. (C) Proteolytic cleavage of PARP. Activation of PARP by proteolytic cleavage was measured in tumor cells treated with or without recombinant human IGFBP3 by Western blot analysis using antibodies for cleaved PARP and β-actin as a loading control. (D) Stable IGFBP3 transfectants. HepT1 cells were transfected with an empty vector control (pIRES) or expression vector containing full-length IGFBP3 cDNA (pIGFBP3), selected with puromycin for 2 weeks and cloned by picking resistant colonies. Exogenous IGFBP3 expression was measured in transfected HepT1 cells using real-time PCR in relation to the house-keeping gene TBP as a calibrator (left) or Western blot analysis using antibodies for IGFBP3 and β-actin as a loading control (right). (E) Growth properties of IGFBP3 transfected HepT1 cells. The viability of stably transfected cells was assessed at the time points indicated using MTT assays and optical density (OD) measurements. The values given represent the mean of triplicate measurements ± SEM. (F) Colony formation assay. HepT1 cells were transfected with an empty vector control (pIRES) or IGFBP3 expression vector (pIGFBP3) and subsequently cultured in puromycin-containing media for 2 weeks. Colonies were stained with crystal violet, and representative assays were photographed (left) and counted (right). Statistically significant difference versus control: *P < 0.05 (unpaired Student's t-test). (G) Annexin V staining. IGFBP3 transfected cells were analyzed for phosphatidylserine membrane asymmetry using Cy5-conjugated annexin V and calcein staining. Representative plots of flow-cytometric measurements depicting 2.4% and 10.6% of apoptotic cells (annexin V and calcein positive) in the control (upper plot) and IGFBP3 transfectants (lower plot) are shown (left). The mean percentages of early apoptotic cells ± SEM of two independent annexin V experiments are given (right). Statistically significant difference versus vehicle: *P < 0.05 (unpaired Student's t-test).