Example 12
Sf9 cells were maintained in Grace's Insect Medium (Invitrogen) plus 10% FBS and split into 48 well plates the day before the transfection at 85% confluence. Cells were transfected with 0.5 μg of dsRNA targeting HSC70 or ATP synthase α and β (SEQ ID NO: 9, 10, and 11, respectively) or GFP (as a negative control) by applying 2.5 μl of TransMessenger reagent (Qiagen) in plain Grace's Insect Medium for 5 hr and replaced the transfection complex with complete Grace's Insect Medium plus 10% FBS for 48 hours according to manufacturer's instructions. Cells were then treated with 200 nM trypsinized Vip3A in plain Grace's Insect Medium without FBS for 48 hours after transfection. Cell viability was measured by adding 50 μl of Cell-Titer Glow solution (Promega) and recording the luminescence with a Luminometer (Beckman). Results are shown in Table 3, expressed as a percent reduction of cell viability.
As shown in Table 2, the toxicity of Vip3 in Sf9 cells with Hsc70 or one or both of ATP synthases α and β is significantly reduced compared to the GFP control, where none of the Vip3 interacting proteins have their expression silenced.