Sgi 7079

Flowcytometric analysis of PD-L1 expression was performed as described previously(25 (link)). Briefly, cells at 70–80% confluency were trypsinized to obtain a single cell suspension. Approximately 500,000 cells were washed with 2% FBS and re-suspended in 100ul 2% FBS and incubated with mouse anti-human PD-L1 (CD274) antibody that was directly conjugated with PE (1:200 dilution, Clone MIH1, BD Pharmingen, San Diego CA) for 30 min at 4°C. Cells were then washed in 2% FBS and analyzed using a Gallios flow cytometer, (Beckman Coulter, Brea CA) and FlowJo software (v.10.1). Prior to collection cells were treated with either Bay 80-6946 (PI3 kinase inhibitor) or SGI-7079 (Axl inhibitor) (both from Selleck Chemical, Houston TX) for 24h where indicated. Immunoblot analysis was performed as described previously(8 ). Commercially available siRNAs specific for either Axl or p110 PI3 kinase (GE Dharmacon, Lafayette CO) were transfected via electroporation (Nucleofector II, Amaxa, Basel, Switzerland) by using program T-001 with transfection agent T (Lonza, Visp, Switzerland). Cells were collected 48–72h post-transfection and assayed as described above. Comparisons between treatment groups were performed using Student’s t-test, with p<0.05 denoting significance.