Figure 4.

Effect of ER stress on gene transfection. (A–C) Results from the test used to determine whether intracellular calcium level changes affected gene transfection. In (A), the cells were pretreated with 10 μM BAPTA-AM, a cell permeable calcium chelating agent, for 30 min, followed by incubation with liposome/DNA complexes for 2 h at 37 °C. In (B), the cells were pretreated with 5 μM or 10 μM BAPTA-AM for 30 min and then transfected with pEGFP. After transfection, the cells were incubated with 5 μM or 10 μM BAPTA-AM for an additional 22 h. In (C), the results from the quantitative analysis of (B) using ImageJ are shown. The scale bar of (A) represents 200 nm, and the scale bar of (B) represents 500 μm. The experiment was done three times. (D) Uptake of the liposomes when the intracellular calcium level was disrupted. Cells were pretreated with 10 μM BAPTA-AM for 30 min, followed by incubation with Lipo-Pars or Lipo-Nons (green) for 2 h at 37 °C. After liposome uptake, the cells were incubated with BAPTA-AM for an additional 1, 4, 10, and 22 h. The scale bar represents 100 μm. (E) Results from experiments used to determine whether ER stress affected gene transfection. Cells were pretreated with BFA (2 μg/mL) and transfected for 2 h. The scale bar represents 500 μm. The experiment was done three times. (F) Results from experiments used to determine how weakened ER stress affected gene transfection. Cells were pretreated with KIRA6 (5 μM), an inhibitor of IRE1α RNase kinase, and transfected for 2 h. The experiment was done three times. (G) Western blot was used to quantify C/EBP-homologous protein (chop/GADD153). Cells were incubated with Lipo-Nons or Lipo-Pars for 2 h. The experiment was done once.