Cytation5 plate

For the micro-injection study, Rockefeller strain Aedes aegypti mosquitoes were injected (100 nL) intrathoracically with virus diluted in PBS so that each mosquito received 50 FFU. Mosquitoes were cultured at 28°C for 8, 12, and 18 days. For blood feeding study, sheep’s blood was centrifuged at 1000 g, 4°C for 20 minutes to separate plasma and cells. The plasma was heat-inactivated at 56°C for 1 hour and the cells were washed twice with PBS after which they were combined again. The blood was spiked with virus to a concentration of 2×10⁶ FFU/mL. Engorged mosquitoes were further reared and harvested at 8, 12, and 18 days. At the time points indicated mosquito samples from both experiments were thoroughly homogenized (Qiagen TissueLyser II) in 200 µL PBS and centrifuged to pellet the tissue. 50 µL of supernatant was used for both RT-qPCR and luciferase assay. RNA was harvested using Qiagen RNeasy minikit and used in a Taqman RT-qPCR reaction targeting a region in NS5. Aedes aegypti actin served as a control. For luciferase assay, 50 µL of supernatant from homogenized mosquito was added to a 96 well opaque white plate, followed by addition of NanoGlo substrate, diluted 1:50 in NanoGlo Assay Buffer. Samples were read in a BioTek Cytation 5 plate reader. Background luciferase levels (no mosquitoes) and clean, uninfected mosquitoes were used as negative controls.

The cells were inoculated in a 96-well plate, an appropriate amount of PBS was added to the wells around the inoculated wells to maintain a suitable humid environment. The abovementioned 96-well plates were placed in a BioTek Cytation5 plate, and the appropriate exposure and length of exposure were set. Shots were taken every 2 h for a total of 132 h of tracking, and the induced cells were cultivated in accordance with the above steps.

Cells were plated in 96-well flat-bottom clear plates at a density of 50,000 cells per well in 100 µL. Cells were exposed to pitavastatin at indicated concentrations, and proliferation was measured 48 h later using a Cell Counting Kit-8 (CCK-8/WST-8) (Dojindo Molecular Technologies, Rockville, MD), following the kit instructions. In this assay, we used 10 μL of the WST-8 reagent, which was added to each well; incubated for 120 min at 37 °C, and then read on the Cytation 5-plate (Bio-Tek) reader using the absorbance at 450 nm. Vehicle treated cells were considered to be controls. The IC₅₀ was determined using a dose–response curve spanning seven log units and calculated using Prism 6 (GraphPad). For the combination treatment studies, we tested whether pretreatment of vincristine resistant REH-VR cells with pitavastatin would augment/sensitize the resistant cells to vincristine. Cells were pretreated with pitavastatin at a concentration of 450 nM for 24 h, after which vincristine (2 nM) was added to the indicated wells, and cell proliferation was measured 48 h later as described, without any media changes.