Day 1: Transfection of RCC4, A498 and 786-O cell lines

Cells were transfected with genome siRNA library (Dharmacon) using Dharmafect3 (RCC4) or LipoRNAiMAXi (A498 and 786-O) and cultured for 1 day.

Day 2: Addition of Everolimus

Everolimus is added on top of the culture media. For control plates, fresh media only was added. The plates were then cultured for further 3 days.

Day 5 Plates are fixed with 80% EthOH and stained with DAPI

Plates were fixed with 80% Ethanol and stained with DAPI.

Readout

Acumen Explorer eX3 laser scanning cytometer was used to determine cell number per well.