After trypsinization, seed approximately 0.25 times 10 to the power of six HEK 293T cells with the transfection mix in a 24-well plate and incubate for six hours at 37 degrees Celsius. Add 125 microliters of complete DMEM medium to the wells and resuspend the cells for even seeding. After 24 hours, replace the existing medium with 250 microliters of incomplete DMEM medium.
Add the freshly prepared second transfection mix and incubate for 36 hours. After centrifugation resuspend the cells in a commercially-procured luciferase substrate. Add the resuspended cell lysate to an opaque 96-well plate and incubate for 10 to 15 minutes.
In luminometer, obtain the luciferase reading. Measure the GFP fluorescence in the same samples to normalize the luciferase reading in a microplate micromode plate reader. Following 24 hours of HEK 293T cells transfection, add one milliliter of complete DMEM to each well of a six-well plate and collect the lentiviral supernatant after 48 hours.
Incubate 2 million Jurkat J6 cells suspended in polybrene containing complete RPMI medium in a six-well plate, with an equal volume of lentiviral supernatant for each control and gene-specific knockdown lentivirus. Add puromycin after 24 hours to each well to select stable cells. Pellet the cells every 24 hours and add two milliliters of fresh complete RPMI 1640 medium with puromycin.
Treat CEM-GFP cells with 100 nanomolar of thapsigargin for 12 hours at 37 degrees Celsius. After washing the cells with RPMI 1640 medium, infect with 0.5 multiplicity of infection of HIV-1. Collect the supernatants from each sample for p24 ELISA.
The cytotoxicity of thapsigargin was analyzed using an MTTSA, showing non-significant cytotoxicity at the desired concentration. Immunoblotting for HSPA5 analyzed the drug's effect on ER stress. Pretreatment with 100 nanomolar thapsigargin for 12 hours induced HSPA5 expression at all time points.
p24 ELIZA showed the effect on virus production.
