G 190

PC12 cells were plated in 96-well plates, serum-deprived for 4 h and subsequently treated with or without NGF (100 ng/mL) or ENT-A010 (500 nM) in the presence or absence of GW441756 TRKA inhibitor (20 μM, G-190, Alomone Labs, Jerusalem, Israel) for another 24 h. The CellTox assay (Promega, Leiden, Belgium) was performed according to the manufacturer’s instructions. Hoechst (1:10,000, Invitrogen, MA, USA) was added for 30 min along with CellTox reagent and cells were imaged with a Zeiss AXIO Vert A1 fluorescent microscope. The number of CellTox⁺ (dead) cells was normalized to the number of Hoechst⁺ cells, the latter depicting the total number of cells, per image. The numbers of CellTox⁺ and Hoechst⁺ cells were determined using the FIJI software.

CellTox assay (G8742, Promega Corporation, Maddison, WI, USA) was used to assess survival of PC12 cells under serum deprivation conditions. PC12 cells were plated in 96-well plates, starved of serum for 4 h and subsequently treated with NGF (100 ng/mL) or ENT-A013 (500 nM) in the presence or absence of TrkA inhibitor GW441756 (20 μM, G-190, Alomone labs, Jerusalem, Israel) for 24 h. CellTox assay reagents and Hoescht (1:10,000, H3570, Invitrogen, Waltham, MA, USA) were added to each well for 30′ and cells were then imaged with a Zeiss AXIO Vert A1 fluorescent microscope (Zeiss, Jena, Germany). CellTox positive cells were normalized to total number of cells for each image.