P21 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.

The human CC cell lines OZ and KKU100 (2 × 10⁵ cells/well) were seeded in 12‐well plates 1 day before transfection. Transfections were performed in Opti‐MEM (ThermoFisher Scientific) using Lipofectamine 3000 (ThermoFisher Scientific) for the transfection of pCMV6‐FLAG‐tagged human SKI expression plasmids (SKI plasmid; OriGene, Rockville, MD, USA) and pCMV6‐empty and Lipofectamine RNAiMAX (ThermoFisher Scientific) for the transfection of human miRNA‐3648 mimic, the miRNA mimic negative control (Dharmacon, Lafayette, CO, USA), SKI siRNA (Abcam, Cambridge, UK), p21 siRNA (Dharmacon), and siCNT (ThermoFisher Scientific). All transfected cells were incubated at 37 °C according to the manufacturer's instructions. The amount of transfected plasmid or RNA used in each well was equalized by the addition of empty plasmid or siCNT, respectively.

Pooled CSE1L siRNA, p21 siRNA (On-target plus smart pool) and Control siRNA (On-target plus siControl non-targeting pool) were purchased from Dharmacon. Cells were transfected according to the manufacturer's guidelines using DharmaFECT I reagent.