BIBF0775 is a selective TGFβ type I receptor (Alk5) inhibitor (IC50: 34 nM).

ALK5:34 nM

The inhibition of the kinase activity of TGFβRI was determined using the Promega Kinase-Glo kit according to the manufacturer's protocol in the presence of 600 nM ATP. N-terminally his-tagged human TGFβRI (aa 162-end) expressed in baculovirus and purified using nickel affinity chromatography was used at a final concentration of 0.03 μg/mL. The inhibition of the kinase activity of PDGFRR was determined using the Z0-LYTE assay technology according to the manufacturer's protocol. Full-length human PDGFRR (5.6 nM per assay) and the Tyr4 peptide (2 μM per assay) were obtained from Invitrogen, too. IC50 values were determined by the use of the Graph PadPrism software.

The high-content cytotoxicity assay (kit I) was performed according to the manufacturer's instructions. HaCaT cells were cultured overnight in black 96-well plates, incubated for 24 h with each compound at different concentrations, and stained with cytotoxicity cocktail. Cells were fixed, washed, and scanned on the Cellomics ArrayScan II platform. Images were analyzed with the Cell Health image analysis algorithm. Cytotoxicity indices were calculated for each of the four parameters (cellular membrane integrity, nuclear fragmentation and density, and lysosomal mass) to indicate the percentage of cells outside of the normal range which was defined using a vehicle-treated reference cell population.