Siglo sirna

The fluorescently labelled siRNA targeting cyclophilin B (siGLO) was selected as this did not influence the phenotype of either EP or DS cultures (Figure S12). HMECs were transfected with 60 nM siGLO siRNA (Dharmacon) or p16 siRNA (Qiagen) in 384‐well plates using Dharmafect 3 (Dharmacon). HMFs were transfected with 30 nM siGLO siRNA or p16 siRNA or p21 siRNA (Dharmacon) in 384‐well plates or 6‐well plates using Dharmafect 2 (Dharmacon). DS + siGLO, DS + p16 siRNA, DS + p21 siRNA or DS + p16 + p21 siRNA cells were harvested for RTqPCR, Western blotting or immunofluorescence as detailed below.

For RNAi, cells (70–80% confluent) were transfected with SMARTpool siRNAs and DharmaFECT 1 transfection reagent (GE Dharmacon) according to manufacturer’s instructions and fixed after 72 h. siGLO siRNA (GE Dharmacon) was used as a control which had no impact on RNA localization. For plasmid transfections, 2–4 μg of DNA was mixed with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions and added to 80–90% confluent cells in a 6-well plate. Cells were fixed and assayed 24, 48, or 72 h after transfection.

A panel of scrambled siRNAs were tested as negative controls in the HMECs, however, each of these induced some degree of toxicity or phenotypic change in the EP cultures. The fluorescently labelled siRNA targeting cyclophilin B (siGLO) was selected for the experiments presented as this did not influence the phenotype of either EP or DS cultures. HMECs were transfected with 60 nM siGLO siRNA (Dharmacon) or p16 siRNA (Qiagen) in 384-well or 6-well plates using Dharmafect3 (Dharmacon). EP+siGLO, DS+siGLO or DS+p16siRNA cells were harvested for flow cytometry, DNA extraction or immunofluorescence as detailed below.