Day 1: Reverse transfection of HaCat cells

HaCaT cells were reverse transfected with genome siRNA (Dharmacon) using INTERFERin transfection reagent in 384 well format.

Day 4: TGF-beta induction and cell fixation

Cells were treated with TGF-beta for 1hour before fixing the cells in plates with 4% formaldehyde for 15-30mins.

Staining

Plates were stained with Smad2/3 anitbody and DAPI.

Readout

• ArrayScan - Smad2/3 nuclear and cytoplasmic intensities (Ch2).
• Acumen Explorer - total cell number per well.