Centricon ym 3

All reagents and PRMI 1640 media were obtained from Sigma–Aldrich Ltd. (Beijing, China) or J&K Scientific Ltd. (Beijing, China). Antioxidant enzyme assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The BCA protein assay kit was obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Polyvinylidine fluoride membranes and Centricon YM-3 were purchased from the Millipore Corp. (USA). Rabbit anti-cyt c (AF0146) and rabbit anti-VDAC1 (DF6140) were obtained from Affinity Biosciences (OH, USA). Fetal bovine serum was purchased from Hangzhou Sijiqing Biomaterials Co., Ltd. (Hangzhou, China).
¹H NMR and ¹³C NMR spectra were recorded using a Varian Mercury spectrometer operating at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR. HR-MS spectra were obtained on an Orbitrap Elite (Thermo Scientific) mass spectrometer. UV/Vis spectra were recorded at 25 °C with a Perkin-Elmer Lambda 25 spectrophotometer that was equipped with temperature-controlled cell holders (USA). Fluorescence intensity and fluorescence spectra were measured at 25 °C with an Infinite M200 Pro Multimode Reader (Switzerland) and a Shimadzu RF-5301 spectrofluorimeter (Japan), respectively.

Western blotting analysis was performed as previously described (17). Briefly, 1×10⁵ PMSCs were transfected with Ad-Endo at an MOI of 3000 (Ad-Endo=3×10⁸ vp, Lipofectamine 2000=1.2 µg). After 48 h, the conditioned media (CM) was harvested and concentrated by ultrafiltration (Centricon YM-3; Millipore, Bedford, MA, USA). Proteins were separated using 12% SDS-PAGE gel and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat milk (Biorad, USA) and then incubated with an anti-endostatin antibody (Santa Cruz Biotechnology, CA) (1:500) followed by horseradish peroxidase-conjugated anti-rabbit immunoglobulins (Sigma-Aldrich, USA) (1:5000). β-actin (1:2000) was used as a loading control, and bands were analyzed using an enhanced chemiluminescence detection system (Thermo Fisher Scientific Inc. USA).