Cerdulatinib

The human beta cell line EndoC-βH1 (kindly provided by Dr. R. Scharfmann, University of Paris, France) was cultured in Matrigel-fibronectin-coated plates as described [4 (link)]. These cells are free from mycoplasma infection, as evaluated by MycoAlert Mycoplasma Detection kit (Lonza, Basel, Switzerland).
Isolation of human islets from 3 non-diabetic organ donors (ESM Table 1) was performed in accordance with the local Ethical Committee in Pisa, Italy. After arrival in Brussels, islets were dispersed and cultured as in [4 (link)]. All experiments shown with EndoC-βH1 cells or human islet cells (indicated as “n” in the figures) refer to independent biological data (i.e using EndoC-βH1 cells from different passages or human islets from different donors). Where indicated, cells were treated with human IFNα (PeproTech Inc., Rocky Hill, NJ) 20 or 1000 U/ml [4 (link)]. Cells were treated with ruxolitinib (kindly provided by Calibr, CA, USA), cerdulatinib (Selleckchem, Germany), Bayer-18 (Synkinase, UK), or cycloheximide (Sigma-Aldrich, Germany) as indicated.

A549 cells, a human alveolar type II like epithelial cell line (American Type Culture Collection, Manassas, VA) and small alveolar epithelial cells (SAECs)(Lonza Inc., San Diego, CA), normal human AECs derived from terminal bronchioli, were grown according to the manufacturer’s instructions. RSV infection in A549 cells were done in F12K medium containing 2% FBS. When SAECs were used for RSV infection, they were changed to basal medium, not supplemented with growth factors, 6 h before and throughout the length of the experiment. At 90 to 95% confluence, cell monolayers were infected with RSV at multiplicity of infection (MOI) of 3. An equivalent amount of 30% sucrose solution was added to uninfected A549 and SAECs, as a control.
For Anacardic acid (10 μM) (172050, EMD Millipore, MA) and Cerdulatinib (5 μM) (PRT062070, Selleckchem, TX) experiments, cells were pretreated with the compounds for 1 h and then infected in their presence for the duration of the experiment. Equal amounts of diluent were added to cells uninfected and infected as control. Total number of cells and cell viability, following various treatments, were measured by trypan blue exclusion. There was no significant change in cell viability with all compounds tested. There was no effect of Anacardic acid on viral replication, while there was a trend in increased viral replication in cells treated with Cerdulatinib.