C6 ceramide

Exosomes and microvesicles were isolated from conditioned media (CM). For CM collection, hADSC were grown to 90% confluence in T75 flasks, media was replaced with serum-free mesenchymal stem cell basal media, with StemFlex medium kit (GibcoLifeTechnologies, Waltham, MA, # A33494-01) and CM was collected after 48 h.
For GW4869-CM, the hADSC cells were treated with GW 4869 (1 µM) (from Cayman Chemical, Ann Arbor, MI, # 13,127) in serum-free media for 48 h. For C2-ceramide CM, hADSC were treated with C2-ceramide (Cayman Chemical, # 62,510), a biologically active, cell permeable and less hydrophobic analog of natural ceramide, at 20 µM in serum-free media, 48 h. For C6-ceramide CM, hADSC were treated with C6-ceramide (Cayman Chemical, # 62,525) at 20 µM with serum-free media, 48 h.

We obtained anti-AR (N-20), anti-BXDC2 (C-1), and anti-GAPDH (6c5) antibodies, and anti-p-ATF2 (Thr71), anti-p-p44/42 MAPK (ERK) (Thr202/Tyr204), anti-PTEN (D4.3), and anti-cleaved caspase-3 (Asp175) antibodies from Santa Cruz Biotechnology and Cell Signaling Technology, respectively. CDDP, DHT, and HF were from Sigma-Aldrich, while MTX, VBL, DOX, GEM, C6 ceramide, and SCH 772984 were from Cayman Chemical.